In vitro evidence that platelet-rich plasma stimulates cellular processes involved in endometrial regeneration.

PURPOSE: The study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endometrial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring. METHODS: Human endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated (a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation (WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR. RESULTS: Activated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Transcripts for inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or progesterone receptor mRNAs was observed. CONCLUSIONS: This is the first study evaluating the effect of PRP on different human endometrial cells involved in tissue regeneration. These data provide an initial ex vivo proof of principle for autologous PRP to promote endometrial regeneration in clinical situations with compromised endometrial growth and scarring.

Stromal fibroblasts from perimenopausal endometrium exhibit a different transcriptome than those from the premenopausal endometrium.

Human endometrium undergoes extensive regeneration on a cyclic basis in premenopausal women and likely occurs through the contribution of stem/progenitor cells. Menopause results in the permanent cessation of menstrual cycles and is preceded by perimenopause, a period of several years in which endocrine and biological changes occur and is a period of risk for endometrial proliferative disorders. The objectives of this study were to identify endometrial mesenchymal stem cells (eMSC) and endometrial stromal fibroblasts (eSF) in endometrium of perimenopausal women and perform expression profile analysis of perimenopausal eMSC and eSF to gain insight into the biology of stem/progenitor and lineage cell populations during the transition to menopause. Endometrial tissue was collected from perimenopausal and premenopausal women (n = 9 each). Microarray analysis was performed on fluorescence-activated cell sorting-isolated eSF and eMSC, and data were validated by quantitative real-time PCR. Principal component analysis showed that cells clustered into three distinct groups in 3-dimensional space: perimenopausal eMSC and premenopausal eMSC clustered together, while perimenopausal eSF and premenopausal eSF formed two discrete clusters separate from eMSC. Hierarchical clustering revealed a branching pattern consistent with principle clustering analysis results, indicating that eMSC from premenopausal and perimenopausal women exhibit similar transcriptomic signatures. Pathway analysis revealed dysregulation of cytoskeleton, proliferation, and survival pathways in perimenopausal vs. premenopausal eSF. These data demonstrate that cell populations have altered gene expression in perimenopausal vs. premenopausal endometrium, and that perimenopausal eSF had altered pathway activation when compared to premenopausal eSF. This study provides insight into aging endometrium with relevance to function in reproductively older women.

Global Transcriptome Abnormalities of the Eutopic Endometrium From Women With Adenomyosis.

OBJECTIVE: Adenomyosis is a clinical disorder defined by the presence of endometrial glands and stroma within the myometrium, the pathogenesis of which is poorly understood. We postulate that dysregulation of genes and pathways in eutopic endometrium may predispose to ectopic implantation. No study, to our knowledge, has examined the global transcriptome of isolated eutopic endometrium from women with clinically significant adenomyosis. DESIGN: Laboratory-based study with full institutional review board approval and consents. MATERIAL AND METHODS: Endometrial sampling was performed on hysterectomy specimens (proliferative phase) from symptomatic women with pathologically confirmed diffuse adenomyosis (n = 3). Controls (n = 5) were normo-ovulatory patients without adenomyosis. All patients were free from leiomyoma, endometriosis, and hormonal exposures. Isolated purified total RNA was subjected to microarray analysis using the Gene 1.0 ST Affymetrix platform. Data were analyzed with GeneSpring and Ingenuity Pathway analysis. Validation of several genes was undertaken by quantitative real-time reverse transcriptase polymerase chain reaction. RESULTS: Comparison of transcriptomes of proliferative endometrium from women with and without adenomyosis revealed 140 upregulated and 884 downregulated genes in samples from women with adenomyosis compared to controls. Highly differentially expressed genes include those involved in regulation of apoptosis, steroid hormone responsiveness, and proteins involved in extracellular matrix remodeling as well as microRNAs of unknown significance. Affected canonical pathways included eukaryotic initiation factor 2 signaling, oxidative phosphorylation, mitochondrial dysfunction, estrogen receptor signaling, and mammalian target of rapamycin signaling. CONCLUSION: The eutopic endometrium in patients with adenomyosis has fundamental abnormalities that may predispose to invasion and survival beyond the myometrial interface.

Human Endometrial Fibroblasts Derived from Mesenchymal Progenitors Inherit Progesterone Resistance and Acquire an Inflammatory Phenotype in the Endometrial Niche in Endometriosis.

Human endometrium undergoes cyclic regeneration involving stem/progenitor cells, but the role of resident endometrial mesenchymal stem cells (eMSC) as progenitors of endometrial stromal fibroblasts (eSF) has not been definitively demonstrated. In endometriosis, eSF display progesterone (P4) resistance with impaired decidualization in vivo and in vitro. To investigate eMSC as precursors of eSF and whether endometriosis P4 resistance is inherited from eMSC, we analyzed transcriptomes of eutopic endometrium eMSC and eSF isolated by fluorescence-activated cell sorting (FACS) from endometriosis (eMSCendo, eSFendo) and controls (eMSCcontrol, eSFcontrol) and their derived primary cultures. Differentially expressed lineage-associated genes (LG) of FACS-isolated eMSC and eSF were largely conserved in endometriosis. In culture, eSFcontrol maintained in vitro expression of a subset of eSF LG and decidualized in vitro with P4 The eMSCcontrol cultures differentiated in vitro to eSF lineage, down-regulating eMSC LG and up-regulating eSF LG, showing minimal transcriptome differences versus eSFcontrol cultures and decidualizing in vitro. Cultured eSFendo displayed less in vitro LG stability and did not decidualize in vitro. In vitro, eMSCendo differentiated to eSF lineage but showed more differentially expressed genes versus eSFendo cultures, and did not decidualize in vitro, demonstrating P4 resistance inherited from eMSCendo Compared to controls, cultures from tissue-derived eSFendo uniquely had a pro-inflammatory phenotype not present in eMSCendo differentiated to eSF in vitro, suggesting divergent niche effects for in vivo versus in vitro lineage differentiation. These findings substantiate eMSC as progenitors of eSF and reveal eSF in endometriosis as having P4 resistance inherited from eMSC and a pro-inflammatory phenotype acquired within the endometrial niche.

Ten new complete mitochondrial genomes of pulmonates (Mollusca: Gastropoda) and their impact on phylogenetic relationships.

BACKGROUND: Reconstructing the higher relationships of pulmonate gastropods has been difficult. The use of morphology is problematic due to high homoplasy. Molecular studies have suffered from low taxon sampling. Forty-eight complete mitochondrial genomes are available for gastropods, ten of which are pulmonates. Here are presented the new complete mitochondrial genomes of the ten following species of pulmonates: Salinator rhamphidia (Amphiboloidea); Auriculinella bidentata, Myosotella myosotis, Ovatella vulcani, and Pedipes pedipes (Ellobiidae); Peronia peronii (Onchidiidae); Siphonaria gigas (Siphonariidae); Succinea putris (Stylommatophora); Trimusculus reticulatus (Trimusculidae); and Rhopalocaulis grandidieri (Veronicellidae). Also, 94 new pulmonate-specific primers across the entire mitochondrial genome are provided, which were designed for amplifying entire mitochondrial genomes through short reactions and closing gaps after shotgun sequencing. RESULTS: The structural features of the 10 new mitochondrial genomes are provided. All genomes share similar gene orders. Phylogenetic analyses were performed including the 10 new genomes and 17 genomes from Genbank (outgroups, opisthobranchs, and other pulmonates). Bayesian Inference and Maximum Likelihood analyses, based on the concatenated amino-acid sequences of the 13 protein-coding genes, produced the same topology. The pulmonates are paraphyletic and basal to the opisthobranchs that are monophyletic at the tip of the tree. Siphonaria, traditionally regarded as a basal pulmonate, is nested within opisthobranchs. Pyramidella, traditionally regarded as a basal (non-euthyneuran) heterobranch, is nested within pulmonates. Several hypotheses are rejected, such as the Systellommatophora, Geophila, and Eupulmonata. The Ellobiidae is polyphyletic, but the false limpet Trimusculus reticulatus is closely related to some ellobiids. CONCLUSIONS: Despite recent efforts for increasing the taxon sampling in euthyneuran (opisthobranchs and pulmonates) molecular phylogenies, several of the deeper nodes are still uncertain, because of low support values as well as some incongruence between analyses based on complete mitochondrial genomes and those based on individual genes (18S, 28S, 16S, CO1). Additional complete genomes are needed for pulmonates (especially for Williamia, Otina, and Smeagol), as well as basal heterobranchs closely related to euthyneurans. Increasing the number of markers for gastropod (and more broadly mollusk) phylogenetics also is necessary in order to resolve some of the deeper nodes -although clearly not an easy task. Step by step, however, new relationships are being unveiled, such as the close relationships between the false limpet Trimusculus and ellobiids, the nesting of pyramidelloids within pulmonates, and the close relationships of Siphonaria to sacoglossan opisthobranchs. The additional genomes presented here show that some species share an identical mitochondrial gene order due to convergence.

Phylogenetic relationships and evolution of pulmonate gastropods (Mollusca): new insights from increased taxon sampling.

Phylogenetic relationships among higher clades of pulmonate gastropods are reconstructed based on a data set including one nuclear marker (complete ribosomal 18S) and two mitochondrial markers (partial ribosomal 16S and Cytochrome oxidase I) for a total of 96 species. Sequences for 66 of these species are new to science, with a special emphasis on sampling the Ellobiidae, Onchidiidae, and Veronicellidae. Important results include the monophyly of Systellommatophora (Onchidiidae and Veronicellidae) as well as the monophyly of Ellobiidae (including Trimusculus, Otina, and Smeagol). Relationships within Ellobiidae, Onchidiidae, and Veronicellidae are evaluated here for the first time using molecular data. Present results are compared with those from the recent literature, and the current knowledge of phylogenetic relationships among pulmonate gastropods is reviewed: despite many efforts, deep nodes are still uncertain. Identification uncertainties about early fossils of pulmonates are reviewed. Impacts of those phylogenetic and fossil record uncertainties on our understanding of the macro-evolutionary history of pulmonates, especially transitions between aquatic and terrestrial habitats, are discussed.