The Impact of Hysteroscopic Tissue Removal Systems on Histopathologic Analysis for Benign and Cancerous Endometrial Pathology: An Ex Vivo Study.

OBJECTIVE: To assess the impact of hysteroscopic tissue removal systems (TRS) on histopathology tissue diagnosis. MEASUREMENTS AND METHODS: This is a paired-comparison ex vivo study in which 23 endometrial sections from hysterectomized uteri (13 benign and 10 hyperplasia/cancer) were analyzed in a simulation laboratory center at a university teaching hospital. After routine tissue processing, a section of endometrium was provided for ex vivo TRS with suture mounting to a uterine model (Polly, Remedy). Morcellated specimens using the Hologic® MyoSure hysteroscopic device were processed for histopathologic analysis by two blinded pathologists (Pa and Pb) and compared to the original specimens' tissue diagnoses. RESULTS: Sufficient tissue for evaluation was found in 100% (23/23) of TRS specimens by Pa and 91.3% by Pb. TRS specimen diagnoses were concordant with routine histologic diagnosis 86.9% (20/23, k = 0.76) for Pa and 80.9% (17/21, k = 0.68) for Pb. Sensitivity and specificity were 70%/100% for Pa and 80%/91% for Pb, respectively. The false-positive (overdiagnosed) and false-negative rates (underdiagnosed) were 0%/30% and 9%/20% for Pa and Pb. Both Pa and Pb underdiagnosed most specimens confirmed by routine tissue diagnosis. TRS specimen diagnoses between Pa and Pb were concordant in 76.2% (16/21, k = 0.60). CONCLUSION: TRS may adversely impact the ability to provide a histologic tissue analysis. Up to 30% of samples were overdiagnosed and 20% underdiagnosed. If confirmed, pathologists may need to reassess workflows to better offset potential underdiagnosis of malignant specimens as findings may be obscured through TRS. Additionally, surgeons may need to reconsider specimen handling, so highest yield specimens are provided to pathology.

Overcoming the Challenging Cervix: Identification and Techniques to Access the Uterine Cavity.

IMPORTANCE: Cervical stenosis is a challenging clinical entity that requires prompt identification and management in order to avoid iatrogenic injury at the time of endocervical canal cannulation. OBJECTIVE: The aim of this study was to identify cervical stenosis and discuss associated etiologies, risk factors, and review medical and surgical approaches for overcoming cervical stenosis. EVIDENCE ACQUISITION: Computerized searches of MEDLINE and PubMed were conducted using the key words "cervix", "cervical stenosis," "embryo transfer," "hysteroscopy complications," "misoprostol," and "ultrasound." References from identified sources were manually searched to allow for a thorough review. Data from relevant sources were compiled to create this review. RESULTS: Transcervical access to the uterine cavity is frequently required for procedures such as hysteroscopy, dilation and curettage, endometrial biopsy, sonohysterogram, hysterosalpingogram, intrauterine insemination, embryo transfer in those undergoing in vitro fertilization, and insertion of intrauterine devices. These procedures can become complicated when difficult cannulation of the endocervical canal is encountered. Management strategies include preprocedural use of cervical-ripening agents or osmotic dilators, ultrasound guidance, no-touch vaginoscopy, manual dilatation, and hysteroscopic resection of the obstructed endocervical canal. CONCLUSIONS AND RELEVANCE: Cervical stenosis is associated with iatrogenic complications that can result in significant patient morbidity. In patients undergoing in vitro fertilization, difficult embryo transfer is associated with lower pregnancy rates. The clinician should carefully consider the patient's menopausal status, risk factors, and symptoms in order to anticipate difficult navigation of the endocervical canal. Various medical and surgical management strategies, including hysteroscopic resection, can be used to overcome the stenotic cervix.

Erythropoietin increases macrophage-mediated T cell suppression.

Erythropoietin (EPO), used to treat anemia in cancer patients, has been reported to accelerate tumor progression and increase mortality. Research of the mechanism for this effect has focused upon EPOR expression by tumor cells. We model the high macrophage to lymphocyte ratio found in tumor microenvironments (TMEs) by culturing peritoneal cavity (PerC) cells that naturally have a high macrophage to T cell ratio. Following TCR ligation, C57BL/6J PerC T cell proliferation is suppressed due to IFNγ-triggered inducible nitric oxide synthase (iNOS) expression. EPO was tested in the PerC culture model and found to increase T cell suppression. This effect could be abrogated by inhibiting iNOS by enzyme inhibition, genetic ablation, or blocking IFNγ signaling. Flow cytometry revealed the EPOR on CD11b(+)F4/80(+) macrophages. These results suggest that EPO could increase T cell suppression in the TME by acting directly on macrophages.

Fetal adrenal capsular cells serve as progenitor cells for steroidogenic and stromal adrenocortical cell lineages in M. musculus.

The lineage relationships of fetal adrenal cells and adrenal capsular cells to the differentiated adrenal cortex are not fully understood. Existing data support a role for each cell type as a progenitor for cells of the adult cortex. This report reveals that subsets of capsular cells are descendants of fetal adrenocortical cells that once expressed Nr5a1. These fetal adrenocortical cell descendants within the adrenal capsule express Gli1, a known marker of progenitors of steroidogenic adrenal cells. The capsule is also populated by cells that express Tcf21, a known inhibitor of Nr5a1 gene expression. We demonstrate that Tcf21-expressing cells give rise to Nr5a1-expressing cells but only before capsular formation. After the capsule has formed, capsular Tcf21-expressing cells give rise only to non-steroidogenic stromal adrenocortical cells, which also express collagen 1a1, desmin and platelet-derived growth factor (alpha polypeptide) but not Nr5a1. These observations integrate prior observations that define two separate origins of adult adrenocortical steroidogenic cells (fetal adrenal cortex and/or the adrenal capsule). Thus, these observations predict a unique temporal and/or spatial role of adult cortical cells that arise directly from either fetal cortical cells or from fetal cortex-derived capsular cells. Last, the data uncover the mechanism by which two populations of fetal cells (fetal cortex derived Gli1-expressing cells and mesenchymal Tcf21-expressing mesenchymal cells) participate in the establishment of the homeostatic capsular progenitor cell niche of the adult cortex.

Genomic markers of ovarian reserve.

Ovarian reserve and its utilization, over a reproductive life span, are determined by genetic, epigenetic, and environmental factors. The establishment of the primordial follicle pool and the rate of primordial follicle activation have been under intense study to determine genetic factors that affect reproductive lifespan. Much has been learned from transgenic animal models about the developmental origins of the primordial follicle pool and mechanisms that lead to primordial follicle activation, folliculogenesis, and the maturation of a single oocyte with each menstrual cycle. Recent genome-wide association studies on the age of human menopause have identified approximately 20 loci, and shown the importance of factors involved in double-strand break repair and immunology. Studies to date from animal models and humans show that many genes determine ovarian aging, and that there is no single dominant allele yet responsible for depletion of the ovarian reserve. Personalized genomic approaches will need to take into account the high degree of genetic heterogeneity, family pedigree, and functional data of the genes critical at various stages of ovarian development to predict women's reproductive life span.

Progression to adrenocortical tumorigenesis in mice and humans through insulin-like growth factor 2 and ß-catenin.

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal ß-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal ß-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized ß-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized ß-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis.

Upstream stimulatory factor induces Nr5a1 and Shbg gene expression during the onset of rat Sertoli cell differentiation.

Within the testis, each Sertoli cell can support a finite number of developing germ cells. During development, the cessation of Sertoli cell proliferation and the onset of differentiation establish the final number of Sertoli cells and, thus, the total number of sperm that can be produced. The upstream stimulatory factors 1 and 2 (USF1 and USF2, respectively) differentially regulate numerous Sertoli cell genes during differentiation. To identify genes that are activated by USF proteins during differentiation, studies were conducted in Sertoli cells isolated from 5- and 11-day-old rats, representing proliferating and differentiating cells, respectively. Usf1 mRNA and USF1 protein levels were increased between 5 and 11 days after birth. In vitro studies revealed that USF1 and USF2 DNA-binding activity also increased at 11 days for the promoters of four potential target genes, Fshr, Gata4, Nr5a1, and Shbg. Chromatin immunoprecipitation assays confirmed that USF recruitment increased in vivo between 5 and 11 days after birth at the Fshr, Gata4, and Nr5a1 promoters. Expression of Nr5a1 and Shbg, but not of Fshr or Gata4, mRNAs was elevated in 11-day-old Sertoli cells compared with 5-day-old Sertoli cells. Transient transfection of USF1 and USF2 expression vectors up-regulated Nr5a1 and Shbg promoter activity. RNA interference assays demonstrated that USF1 and USF2 contribute to Nr5a1 and Shbg expression in differentiating cells. Together, these data indicate that increased USF levels induce the expression of Nr5a1 and Shbg during the differentiation of Sertoli cells, whereas Fshr and Gata4 expression is not altered by USF proteins during differentiation.

Adrenocortical stem and progenitor cells: unifying model of two proposed origins.

The origins of our understanding of the cellular and molecular mechanisms by which signaling pathways and downstream transcription factors coordinate the specification of adrenocortical cells within the adrenal gland have arisen from studies on the role of Sf1 in steroidogenesis and adrenal development initiated 20 years ago in the laboratory of Dr. Keith Parker. Adrenocortical stem/progenitor cells have been predicted to be undifferentiated and quiescent cells that remain at the periphery of the cortex until needed to replenish the organ, at which time they undergo proliferation and terminal differentiation. Identification of these stem/progenitor cells has only recently been explored. Recent efforts have examined signaling molecules, including Wnt, Shh, and Dax1, which may coordinate intricate lineage and signaling relationships between the adrenal capsule (stem cell niche) and underlying cortex (progenitor cell pool) to maintain organ homeostasis in the adrenal gland.

Follicle-stimulating hormone (FSH) transiently blocks FSH receptor transcription by increasing inhibitor of deoxyribonucleic acid binding/differentiation-2 and decreasing upstream stimulatory factor expression in rat Sertoli cells.

FSH acts through the FSH receptor (FSHR) to modulate cell processes that are required to support developing spermatozoa. Within the testis, only Sertoli cells possess receptors for FSH and are the major targets for this regulator of spermatogenesis. FSH stimulation of Sertoli cells for 24-48 h is known to induce Fshr mRNA expression through an E-box motif (CACGTG) located 25 bp upstream of the transcription start site. In contrast, FSH stimulation for 8 h inhibits Fshr transcription. DNA-protein binding studies performed using nuclear extracts from Sertoli cells show that protein binding to the Fshr promoter E-box was reduced 68% after 6 h of FSH stimulation but increased 191% over basal levels after 48 h of stimulation. The proteins binding to the Fshr E-box were identified as upstream stimulatory factor (USF)-1 and -2. FSH stimulation transiently decreased USF1 levels and increased the expression of the inhibitor of DNA binding/differentiation (ID)-2 repressor protein with the same kinetics as the decreased USF/E-box interactions. Overexpression of ID2 resulted in a dose-dependent decrease in USF-driven Fshr promoter activity in the MSC-1 Sertoli cell line, and ID2 inhibited USF binding to the Fshr E-box. Together, these studies suggest that stimulation of Sertoli cells with FSH transiently decreases expression of the USF1 activator and induces accumulation of the ID2 repressor, to block USF binding to the Fshr promoter and delay activation of Fshr transcription. This FSH-regulated mechanism may explain the cyclical changes in Fshr expression that occurs in Sertoli cells in vivo.

USF1/2 transcription factor DNA-binding activity is induced during rat Sertoli cell differentiation.

Each Sertoli cell can support a finite number of developing germ cells. During development of the testis, the cessation of Sertoli cell proliferation and the onset of differentiation determine the final number of Sertoli cells and, hence, the number of sperm that can be produced. We hypothesize that the transition from proliferation to differentiation is facilitated by E-box transcription factors that induce the expression of differentiation-promoting genes. The relative activities of E-box proteins were studied in primary Sertoli cells isolated from 5-, 11-, and 20-day-old rats, representing proliferating, differentiating, and differentiated cells, respectively. E-box DNA-binding activity is almost undetectable 5 days after birth but peaks with initiation of differentiation 11 days after birth and remains elevated. Upstream stimulatory factors 1 and 2 (USF1 and USF2) were found to be the predominant E-box proteins present within DNA-protein complexes formed after incubating E-box-containing probes with nuclear extracts from developing Sertoli cells. The known potentiator of Sertoli cell differentiation, thyroxine, increases USF DNA-binding activity in Sertoli cells before differentiation (5-day-old Sertoli cells) but not after differentiation is initiated (11- and 20-day-old Sertoli cells). The developmental-specific increase in USF1 and USF2 DNA-binding activity may facilitate the switch from proliferation to differentiation and, thus, determine the ultimate number of Sertoli cells present within the testes and the upper limit of fertility.

CCR7 mediates inflammation-associated tumor progression.

Chemokine receptor 7 (CCR7) mediates leukocyte adhesion and chemotaxis from peripheral sites of inflammation through lymphatic channels to secondary lymphoid organs. Aberrant CCR7 expression has been identified on certain tumor types and been linked to pro-survival and invasive pathways. In metastatic squamous cell carcinoma of the head and neck (SCCHN), we have described the selective upregulation of functional CCR7. In this manuscript, we review our understanding of CCR7-mediated signaling in metastatic SCCHN and provide evidence for its involvement in tumor survival, invasion, and metastasis. Autocrine and paracrine CCR7 activation appears to propagate the response to the CCR7 ligands CCL19 and CCL21, which are expressed by the lymphatic endothelium, secondary lymphoid tissues, and CCR7-positive tumor cells. Based on our recent findings, the induction of CCR7 expression and the sustenance of the autocrine signaling pathway have been shown to be regulated by NF-kappaB, similar to several types of immune cells. While extending these observations to metastatic SCCHN tumor cells, our studies highlight the importance of downstream NF-kappaB mediated CCR7 signals in the progression of SCCHN malignancy.