Successful implantation is the result of reciprocal interactions between the implantation-competent blastocyst and receptive uterus. Although various cellular aspects and molecular pathways of this dialogue have been identified, a comprehensive understanding of the implantation process is still missing. The receptive state of the uterus, which lasts for a limited period, is defined as the time when the uterine environment is conducive to blastocyst acceptance and implantation. A better understanding of the molecular signals that regulate uterine receptivity and implantation competency of the blastocyst is of clinical relevance because unraveling the nature of these signals may lead to strategies to correct implantation failure and improve pregnancy rates. Gene expression studies and genetically engineered mouse models have provided valuable clues to the implantation process with respect to specific growth factors, cytokines, lipid mediators, adhesion molecules, and transcription factors. However, a staggering amount of information from microarray experiments is also being generated at a rapid pace. If properly annotated and explored, this information will expand our knowledge regarding yet-to-be-identified unique, complementary, and/or redundant molecular pathways in implantation. It is hoped that the forthcoming information will generate new ideas and concepts for a process that is essential for maintaining procreation and solving major reproductive health issues in women.

Molecular cues to implantation

High density cDNA microarray screening was used to determine changes in gene expression occurring during the transition between the early luteal (prereceptive) and mid-luteal (receptive) phases in human endometrium. Of approximately 12,000 genes profiled, 693 (5.8%) displayed >2-fold differences in relative levels of expression between these stages. Of these, 370 genes (3.1%) displayed decreases ranging from 2- to >100-fold while 323 genes (2.7%) displayed increases ranging from 2- to >45-fold. Many genes correspond to mRNAs encoding proteins previously shown to change in a similar manner between the proliferative and mid-luteal phases, serving as one validation of the microarray screening results. In addition, novel genes were identified. Genes encoding cell surface receptors, adhesion and extracellular matrix proteins and growth factors accounted for 20% of the changes. Several genes were studied further by Northern blot analyses. These results confirmed that claudin-4/Clostridium perfringens enterotoxin (CPE) receptor and osteopontin (OPN) mRNA increased approximately 4- and 12-fold respectively, while betaig-H3 (BIGH3) decreased >80% during the early to mid-luteal transition. Immunostaining also revealed strong specific staining for claudin-4/CPE, EP(1) and prostaglandin receptor in epithelia, and leukotriene B4 receptor in both epithelia and stroma, at the mid-luteal stage. Collectively, these studies identify multiple new candidate markers that may be used to predict the receptive phase in humans. Some of these gene products, e.g. OPN, may play direct roles in embryo-uterine interactions during the implantation process.

/pdf/changes-in-gene-expression-during-the-early-to-mid-luteal-4u7vgv6i60.pdf

Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening.

Regulators of the cell cycle such as cyclin E play an important part in neoplasia. The cyclin E protein forms a partnership with a specific protein kinase. This complex phosphorylates key substrates to initiate DNA synthesis. Cyclin-dependent kinase inhibitors (CKIs) are able to suppress the activity of cyclin E. Various substances (including proteins produced by oncogenic viruses) affect cyclin E directly or indirectly through an interaction with CKIs. These interactions are important in elucidating the mechanisms of neoplasia. They may also provide prognostic information in a wide range of common cancers. Cyclin E may even be a target for treatment of cancers in the future.

Cyclin E in human cancers

Endometriosis is a common disease seen by gynecologists. Clinical features involve pelvic pain and unexplained infertility. Although endometriosis is pathologically characterized by endometrial tissue outside the normal uterine location, endometriosis is otherwise not easily explained. Endometriomas, endometriotic cysts of the ovary, typically cause pain and distortion of pelvic anatomy. To begin to understand the pathogenesis of endometriomas, we describe the first transcriptome-microRNAome analysis of endometriomas and eutopic endometrium using next-generation sequencing technology. Using this approach, we generated a total of more than 54 million independent small RNA reads from our 19 clinical samples. At the microRNA level, we found 10 microRNA that were up-regulated (miR-202, 193a-3p, 29c, 708, 509-3-5p, 574-3p, 193a-5p, 485-3p, 100, and 720) and 12 microRNA that were down-regulated (miR-504, 141, 429, 203, 10a, 200b, 873, 200c, 200a, 449b, 375, and 34c-5p) in endometriomas compared with endometrium. Using in silico prediction algorithms, we correlated these microRNA with their corresponding differentially expressed mRNA targets. To validate the functional roles of microRNA, we manipulated levels of miR-29c in an in vitro system of primary cultures of human endometrial stromal fibroblasts. Extracellular matrix genes that were potential targets of miR-29c in silico were significantly down-regulated using this biological in vitro system. In vitro functional studies using luciferase reporter constructs further confirmed that miR-29c directly affects specific extracellular matrix genes that are dysregulated in endometriomas. Thus, miR-29c and other abnormally regulated microRNA appear to play important roles in the pathophysiology of uterine function and dysfunction.

Functional microRNA involved in endometriosis.

Immunocytochemistry of oestrogen receptor (ER) and progesterone receptor (PR) expression of the whole uterine muscular wall and the endometrium was performed in order to obtain morphological and functional insights into the regulation of cyclic uterine peristalsis, which is confined to the endometrium and the subendometrial myometrium and serves functions such as rapid and sustained sperm transport. The study revealed that the subendometrial myometrium or stratum subvasculare with a predominantly circular arrangement of muscular fibres exhibits a cyclic pattern of ER and PR expression that parallels that of the endometrium, whereas the outer portion of the uterine wall composed of the stratum vasculare and supravasculare, which represents the bulk of the uterine musculature, does not exhibit a cyclic pattern of ER and PR expression. According to ontogenetic and phylogenetic data from the literature, the outer myometrium is of non-paramesonephric origin with functions confined to parturition, while the inner myometrial layer together with the glandular epithelium and the stroma of the endometrium is of paramesonephric origin with various functions during the cycle in addition to those during pregnancy and parturition. The inner quarter of the stratum vasculare adjacent to the stratum subvasculare constitutes a transitional zone in that the cyclicity of receptor staining becomes, in radial direction, gradually less expressed. Morphologically this zone corresponds to the inner part of the stratum vasculare where its muscular fibres blend with those of the stratum subvasculare.

/pdf/the-cyclic-pattern-of-the-immunocytochemical-expression-of-15wx59xsrf.pdf

The cyclic pattern of the immunocytochemical expression of oestrogen and progesterone receptors in human myometrial and endometrial layers: characterization of the endometrial–subendometrial unit

Cell cycle regulatory molecules were analysed in normal human endometrial tissue using antibodies against cyclins D1, E, A, and B1 and cyclin-dependent kinases (CDKs) cdk4, cdk2, and cdc2. The expression of these regulatory molecules in gland cells and stromal cells was compared with the expression of oestrogen receptors (ER), progesterone receptors (PR), and Ki67 (a growth-related molecule). In general, a substantially higher percentage of the gland cells stained positive for cyclins and CDKs during the proliferative phase of the menstrual cycle. Cyclin E, cdk2 and/or cdk4 were especially apparent in the cytoplasm of most of the gland cells as well as in the stromal cells. In contrast, most of the regulatory molecules were undetectable in the gland cells by the end of the secretory phase of the cycle, but they did not decline in the stromal cells. The data also revealed that ER, PR, and Ki67 in both gland cells and stromal cells follow the same basic pattern of expression as the cyclins and CDKs. These results suggest that cyclins and CDKs are functionally involved in the rhythmic proliferation of normal human endometrial tissue, and the action of these agents may be related to the endometrial levels of sex steroids and Ki67.

/pdf/relationship-between-the-expression-of-cyclins-cyclin-2mbj4poyz6.pdf

Relationship between the expression of cyclins/cyclin-dependent kinases and sex-steroid receptors/Ki67 in normal human endometrial glands and stroma during the menstrual cycle

Shuan-fung Li

Papers

Relationship between the expression of cyclins/cyclin-dependent kinases and sex-steroid receptors/Ki67 in normal human endometrial glands and stroma during the menstrual cycle