Anne M. Macpherson

Cell proliferation is increased in the endometrium of women with endometriosis

OBJECTIVE To compare the proliferation of endothelial, epithelial, and stromal cells in the endometrium of women with endometriosis and normal controls. DESIGN Proliferating cells were identified using the monoclonal antibody antiproliferating cell nuclear antigen. A second antibody (CD34) was used to identify endothelial cells (ECs). SETTING University Department of Obstetrics and Gynaecology. PATIENTS Women with laparoscopically proven endometriosis, n = 30. Controls were women with a normal pelvis at laparoscopy performed for tubal sterilization or for infertility due to a male factor, n = 27. MAIN OUTCOME MEASURES Endothelial cells: proliferative index. Epithelial and stromal cells: semi-quantitative immunostaining score. RESULTS The mean EC proliferative index was significantly greater in those with endometriosis compared with controls. This difference was most marked during the proliferative phase of the menstrual cycle. Proliferative phase epithelial and stromal cells demonstrated significantly higher immunostaining scores in endometriosis patients than in controls. CONCLUSIONS We have demonstrated increased numbers of proliferating ECs as well as epithelial and stromal cells in proliferative phase endometrium of women with endometriosis. This suggests that the endometrium of these women might have an enhanced ability to implant and survive in ectopic locations.

TGF-β Mediates Proinflammatory Seminal Fluid Signaling in Human Cervical Epithelial Cells

The cervix is central to the female genital tract immune response to pathogens and foreign male Ags introduced at coitus. Seminal fluid profoundly influences cervical immune function, inducing proinflammatory cytokine synthesis and leukocyte recruitment. In this study, human Ect1 cervical epithelial cells and primary cervical cells were used to investigate agents in human seminal plasma that induce a proinflammatory response. TGF-β1, TGF-β2, and TGF-β3 are abundant in seminal plasma, and Affymetrix microarray revealed that TGF-β3 elicits changes in Ect1 cell expression of several proinflammatory cytokine and chemokine genes, replicating principal aspects of the Ect1 response to seminal plasma. The differentially expressed genes included several induced in the physiological response of the cervix to seminal fluid in vivo. Notably, all three TGF-β isoforms showed comparable ability to induce Ect1 cell expression of mRNA and protein for GM-CSF and IL-6, and TGF-β induced a similar IL-6 and GM-CSF response in primary cervical epithelial cells. TGF-β neutralizing Abs, receptor antagonists, and signaling inhibitors ablated seminal plasma induction of GM-CSF and IL-6, but did not alter IL-8, CCL2 (MCP-1), CCL20 (MIP-3α), or IL-1α production. Several other cytokines present in seminal plasma did not elicit Ect1 cell responses. These data identify all three TGF-β isoforms as key agents in seminal plasma that signal induction of proinflammatory cytokine synthesis in cervical cells. Our findings suggest that TGF-β in the male partner’s seminal fluid may influence cervical immune function after coitus in women, and potentially be a determinant of fertility, as well as defense from infection.

Immunolocalization of the vasoconstrictor endothelin in human endometrium during the menstrual cycle and in umbilical cord at birth

OBJECTIVE Our objective was to determine the localization of immunoreactive endothelin in human cyclic endometrium and in umbilical cord during normal delivery and after cesarean section. STUDY DESIGN Fixed dated endometrial tissue (n = 41) and umbilical cord (n = 6) were subjected to immunohistochemistry with an antiserum cross reacting with endothelin-1, -2 and -3. RESULTS Low levels of stromal endometrial staining were seen throughout the cycle. The strongest staining was in luminal epithelium throughout the secretory phase and in glandular epithelium in the late-secretory phase. In umbilical cord the most intense immunoreactivity was present on the amnion cells on the outer cord, with some staining in intermittent cells in the Whartons jelly and in umbilical vein cells. No differences were detected between cord from normal delivery or cesarean section. CONCLUSION A paracrine role is suggested for endothelin in regulation of endometrial function and a role in vasoconstriction in the umbilical cord at birth.

Stress response genes are suppressed in mouse preimplantation embryos by granulocyte-macrophage colony-stimulating factor (GM-CSF)

BACKGROUND Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to promote the development and survival of human and mouse preimplantation embryos; however, the mechanism of action of GM-CSF in embryos is not defined. METHODS Mouse blastocysts were cultured from zygote stage in vitro with and without recombinant mouse GM-CSF (rmGM-CSF), and in vivo developed blastocysts were flushed from Csf2 null mutant and wild-type mice. The effect of GM-CSF on blastocyst expression of stress response and apoptosis genes was evaluated by microarray, qPCR and immunochemistry. RESULTS Microarray analysis of the gene transcription profile showed suppression of stress response and apoptosis gene pathways in blastocysts exposed to rmGM-CSF in vitro. qPCR analysis confirmed that rmGM-CSF inhibited expression of heat shock protein (HSP) and apoptosis pathway genes Cbl, Hspa5, Hsp90aa1, Hsp90ab1 and Gas5 in in vitro blastocysts. Immunocytochemical analysis of HSP 1 (HSPA1A/1B; HSP70), BAX, BCL2 and TRP53 (p53) in in vitro blastocysts showed that HSPA1A/1B and BCL2 proteins were less abundant when embryos were cultured with rmGM-CSF. BAX and TRP53 were unchanged at the protein level, but Bax mRNA expression was reduced after GM-CSF treatment. In in vivo developed blastocysts, Csf2 null mutation caused elevated expression of Hsph1 but not other stress response genes. CONCLUSIONS We conclude that GM-CSF inhibits the cellular stress response and apoptosis pathways to facilitate embryo growth and survival, and the protective effects of GM-CSF are particularly evident in in vitro culture media, whereas in vivo other cytokines can partly compensate for absence of GM-CSF.

Csf2 Null Mutation Alters Placental Gene Expression and Trophoblast Glycogen Cell and Giant Cell Abundance in Mice

Abstract Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31+ glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C+, P57Kip2+) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Microarray analysis of mRNA from cumulus cells following in vivo or in vitro maturation of mouse cumulus–oocyte complexes

The IVM of mammalian cumulus-oocyte complexes (COCs) yields reduced oocyte developmental competence compared with oocytes matured in vivo. Altered cumulus cell function during IVM is implicated as one cause for this difference. We have conducted a microarray analysis of cumulus cell mRNA following IVM or in vivo maturation (IVV). Mouse COCs were sourced from ovaries of 21-day-old CBAB6F1 mice 46h after equine chorionic gonadotrophin (5IU, i.p.) or from oviducts following treatment with 5IU eCG (61h) and 5IU human chorionic gonadotrophin (13h). IVM was performed in α-Minimal Essential Medium with 50 mIU FSH for 17h. Three independent RNA samples were assessed using the Affymetrix Gene Chip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA, USA). In total, 1593 genes were differentially expressed, with 811 genes upregulated and 782 genes downregulated in IVM compared with IVV cumulus cells; selected genes were validated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Surprisingly, haemoglobin α (Hba-a1) was highly expressed in IVV relative to IVM cumulus cells, which was verified by both RT-PCR and western blot analysis. Because haemoglobin regulates O2 and/or nitric oxide availability, we postulate that it may contribute to regulation of these gases during the ovulatory period in vivo. These data will provide a useful resource to determine differences in cumulus cell function that are possibly linked to oocyte competence.

Surface ultrastructure of uterine epithelial cells in women with premature ovarian failure following steroid hormone replacement.

Scanning electron microscopy has been used to study the apical surface of uterine epithelial cells in women with premature ovarian failure following steroid hormone replacement therapy. A variety of ultrastructural characteristics are identified which could indicate a uterus that is receptive for blastocyst implantation.

Obesity alone or with type 2 diabetes is associated with tissue specific alterations in DNA methylation and gene expression of PPARGC1A and IGF2

Abstract Background: Epigenetic modifications of key genes have been linked to the development of aging related diseases, such as type 2 diabetes, with increased DNA methylation of the transcriptional co-activator,

Ceruloplasmin gene expression in the rat uterus

In this paper we identify the uterine epithelial cells as the site of ceruloplasmin gene expression in the uterus. We show that during the estrous cycle ceruloplasmin RNA levels change substantially reaching a maximum at estrous. In the absence of endocrine control by the ovaries, ceruloplasmin RNA is still present suggesting that this gene is constitutively expressed by the uterine epithelial cells. However, the changes in RNA level immediately after ovariectomy and during pregnancy suggest that it is also the subject of a complex regulatory system.

Ultrastructural Study of Human Uterine Epithelium from a Patient with a Confirmed Pregnancy

Scanning and transmission electron microscopy have been used to study the uterine epithelial cells from a pregnant human uterus approximately 8 days after ovulation. The ultrastructural appearance of the epithelial cells generally conforms with that previously described as showing receptivity, although some significant regional variability exists.