Transanal total mesorectal excision for rectal cancer: state of the art.

Achieving a high-quality total mesorectal excision (TME) resection specimen is a central tenet of curative rectal cancer management. However, operating at the caudal extremity of the pelvis is inherently challenging and a number of patient- and tumour-related factors may increase the risk of obtaining a poor TME specimen and positive resection margins. Transanal TME (TaTME) is an advanced surgical technique developed to overcome the limitations in pelvic exposure and instrumentation of transabdominal surgery. This up-to-date narrative review describes the evolution of TME surgery, the indications for TaTME, current published outcomes, its limitations and future developments.

Robotic transanal minimally invasive surgery (TAMIS) for local excision of rectal lesions with the da Vinci Xi (dVXi): technical considerations and video vignette.

Robotic transanal minimally invasive surgery (TAMIS) (RT) represents a compelling new alternative capable of overcoming the limitations of conventional TAMIS for the local excision of rectal lesions. We describe our RT technique using the dVXi™ (Intuitive Surgical, Sunnyvale, CA, USA) which we have used to efficiently and completely excise eight cases of rectal lesions which were not endoscopically resectable. We also include a video vignette of the procedure. With the patient in the prone jackknife position, we insert a GelPOINT™ Path Transanal Access Platform (Applied Medical, Rancho Santa Margarita, CA, USA) in combination with the dVXi and AirSeal™ insufflation system (Conmed, Niagara. Falls, ON, Canada). Our technique aims to be ergonomically efficient to minimise docking difficulties and to reduce instrument clash in the limited space, whilst maximising the capabilities of the dVXi for RT. At 3-month endoscopic follow-up, no evidence of recurrence was detected in any of the eight patients. RT is safe, feasible and has advantages over conventional laparoscopic TAMIS (LT). Our described technique addresses some of the long-standing challenges of LT and the novel RT. The immediate challenge to its widespread use remains the cost, expertise and availability.

Indocyanine green fluorescence angiography (ICGFA) during laparoscopic and robotic colorectal surgery- a video vignette.

Anastomotic leaks are a dreaded complication of all colorectal surgery with the main factors contributing to it being tension on the anastomosis, intra-abdominal or systemic sepsis, distal obstruction, inadequate blood supply and improper surgical techniques. The leak rate of left-sided high colorectal resections can have a clinically significant leak rate from as low as 1-5% in high anterior resections to 7.9% in low anastomoses. This article is protected by copyright. All rights reserved.

The human oviduct transcriptome reveals an anti-inflammatory, anti-angiogenic, secretory and matrix-stable environment during embryo transit.

The human oviduct serves as a conduit for spermatozoa in the peri-ovulatory phase and nurtures and facilitates transport of the developing embryo for nidation during the luteal phase. Interactions between the embryo and oviductal epithelial surface proteins and secreted products during embryo transit are largely undefined. This study investigated gene expression in the human oviduct in the early luteal versus follicular phases to identify candidate genes and biomolecular processes that may participate in maturation and transport of the embryo as it traverses this tissue. Oviductal RNA was hybridized to oligonucleotide arrays and resulting data were analysed by bioinformatic approaches. There were 650 genes significantly down-regulated and 683 genes significantly up-regulated (P<0.05) in the luteal versus follicular phase. Quantitative real-time PCR, immunoblot analysis and immunohistochemistry confirmed selected gene expression and cellular protein localization. Down-regulated genes involved macrophage recruitment, immunomodulation and matrix-degeneration, and up-regulated genes involved anti-inflammatory, ion transport, anti-angiogenic and early pregnancy recognition. The oviduct displayed some similarities and differences in progesterone-regulated genes compared with the human endometrium. Together, these data suggest a unique hormonally regulated environment during embryo development, maturation and transport through human oviduct and some conservation of progesterone signalling in tissues of common embryological origin. The oviduct serves as a conduit for spermatozoa in the peri-ovulatory phase and it nurtures and facilitates transport of the developing embryo during the luteal phase of the menstrual cycle, although precise interactions between the embryo and oviductal epithelium and secreted products are largely undefined. Herein, we investigated gene expression in human oviduct to identify candidate genes and processes that may participate in maturation and transport of the embryo as it develops implantation competence. Total RNA from human ampullary oviducts in the early luteal versus follicular phases was isolated and hybridized to oligonucleotide arrays. The data, analysed by bioinformatic approaches, revealed that 650 genes were significantly down- and 683 genes were significantly up-regulated in the luteal phase. Quantitative real-time PCR, immunoblot analysis and immunohistochemistry confirmed selected gene expression and cellular protein localization. The data demonstrated down-regulation of genes involved in macrophage recruitment, immunomodulation and matrix degeneration and up-regulation of ion transport and secretions, as well as anti-angiogenic and early pregnancy recognition. Together, these data suggest a unique hormonally regulated environment during embryo development, maturation and transport through the human oviduct and provide insight into mechanisms influencing acquisition of implantation competence of the human embryo during its passage through the oviduct en route to the uterine endometrium.

Closure of the open abdomen.

The open abdomen is a valuable tool in the management of patients with intra-abdominal hypertension and abdominal compartment syndrome. The longer an abdomen is left open, the greater the potential morbidity, however. From the very start, specific measures should be considered to increase the likelihood of definitive closure and prevent the development of visceral adhesions, lateralization, and/or loss of skin and fascia, ileus, fistulae, and malnutrition. Early definitive closure of all abdominal wall layers is the short-term goal of management once the need for the open abdomen has resolved. Several devices and strategies improve the chances for definitive closure. If a frozen abdomen develops, split-thickness skin grafting of a granulating open abdominal wound base is an alternative. Early coverage of the exposed viscera and acceptance of a large abdominal hernia permit earlier reversal of the catabolic state and lower the risk of fistula formation. When a stoma is required, sealing and separation can become problematic. If a fistula develops, a more complex situation prevails, requiring specific techniques to isolate its output and a longer-term strategy to restore intestinal continuity. Planning the closure of an open abdomen is a process that starts on the first day that the abdomen is opened. Multiple factors need to be addressed, optimized, and controlled to achieve the best outcome.

MicroRNA expression profiling of eutopic secretory endometrium in women with versus without endometriosis.

Endometriosis is a common gynecologic disorder characterized by pain and infertility. In addition to estrogen dependence, progesterone resistance is an emerging feature of this disorder. Specifically, a delayed transition from the proliferative to secretory phase as evidenced by dysregulation of progesterone target genes and maintenance of a proliferative molecular fingerprint in the early secretory endometrium (ESE) has been reported. MicroRNAs (miRNAs) are small noncoding RNAs that collectively represent a novel class of regulators of gene expression. In an effort to investigate further the observed progesterone resistance in the ESE of women with endometriosis, we conducted array-based, global miRNA profiling. We report distinct miRNA expression profiles in the ESE of women with versus without endometriosis in a subset of samples previously used in global gene expression analysis. Specifically, the miR-9 and miR-34 miRNA families evidenced dysregulation. Integration of the miRNA and gene expression profiles provides unique insights into the molecular basis of this enigmatic disorder and, possibly, the regulation of the proliferative phenotype during the early secretory phase of the menstrual cycle in affected women.

Expression of membrane progesterone receptors on human T lymphocytes and Jurkat cells and activation of G-proteins by progesterone.

Although there is significant evidence for progesterone's role as an immunomodulator, nuclear progesterone receptors have not been consistently identified in immune cells. Recently, three new putative membrane progesterone receptors (mPRs), mPRalpha, mPRbeta, and mPRgamma have been described. The objective of this study was to examine whether mPRs are expressed in peripheral blood leukocytes (PBLs) in women of reproductive age, and to further characterize them in T lymphocytes and immortalized T cells (Jurkat cells). Transcripts for mPRalpha and mPRbeta but not mPRgamma, were detected by RT-PCR in PBLs, T lymphocytes, and Jurkat cells. Western blot analysis showed the presence of the mPRalpha and mPRbeta proteins on cell membranes of T lymphocytes and Jurkat cells. Expression of the mPRalpha mRNA was upregulated in the luteal phase of the menstrual cycle in cluster of differentiation (CD)8+, but not in CD4+, T lymphocytes. Radioreceptor assays revealed specific [(3)H]progesterone binding to T- and Jurkat cell membranes (K(d) 4.25 nM) characteristic of steroid membrane receptors. Progesterone activated an inhibitory G-protein (G(i)), suggesting that mPRs are coupled to G(i) in Jurkat cells. These results suggest a potential novel mechanism for progesterone's immunoregulatory function through activation of mPRs.

Uterine receptivity to human embryonic implantation: histology, biomarkers, and transcriptomics.

Embryonic implantation is a dynamic process of paracrine interactions between the maternal compartment and the conceptus and involves a receptive endometrium and a developmentally competent blastocyst. Herein, we review histology, clinical approaches, and the promise of transcriptomics in elucidating mechanisms underlying implantation and development of biomarkers of uterine receptivity-with an eye to diagnose and treat implantation-based disorders of miscarriage, fetal growth restriction, pre-eclampsia, and infertility.

Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators.

During the invasive phase of implantation, trophoblasts and maternal decidual stromal cells secrete products that regulate trophoblast differentiation and migration into the maternal endometrium. Paracrine interactions between the extravillous trophoblast and the maternal decidua are important for successful embryonic implantation, including establishing the placental vasculature, anchoring the placenta to the uterine wall, and promoting the immunoacceptance of the fetal allograph. To our knowledge, global crosstalk between the trophoblast and the decidua has not been elucidated to date, and the present study used a functional genomics approach to investigate these paracrine interactions. Human endometrial stromal cells were decidualized with progesterone and further treated with conditioned media from human trophoblasts (TCM) or, as a control, with control conditioned media (CCM) from nondecidualized stromal cells for 0, 3, and 12 h. Total RNA was isolated and processed for analysis on whole-genome, high-density oligonucleotide arrays containing 54,600 genes. We found that 1374 genes were significantly upregulated and that 3443 genes were significantly downregulated after 12 h of coincubation of stromal cells with TCM, compared to CCM. Among the most upregulated genes were the chemokines CXCL1 (GRO1) and IL8,CXCR4, and other genes involved in the immune response (CCL8 [SCYA8], pentraxin 3 (PTX3), IL6, and interferon-regulated and -related genes) as well as TNFAIP6 (tumor necrosis factor alpha-induced protein 6) and metalloproteinases (MMP1, MMP10, and MMP14). Among the downregulated genes were growth factors, e.g., IGF1, FGF1, TGFB1, and angiopoietin-1, and genes involved in Wnt signaling (WNT4 and FZD). Real-time RT-PCR and ELISAs, as well as immunohistochemical analysis of human placental bed specimens, confirmed these data for representative genes of both up- and downregulated groups. The data demonstrate a significant induction of proinflammatory cytokines and chemokines, as well as angiogenic/static factors in decidualized endometrial stromal cells in response to trophoblast-secreted products. The data suggest that the trophoblast acts to alter the local immune environment of the decidua to facilitate the process of implantation and ensure an enriched cytokine/chemokine environment while limiting the mitotic activity of the stromal cells during the invasive phase of implantation.

Angiopoietin-1 and -2 mRNA and protein expression in mouse preimplantation embryos and uteri suggests a role in angiogenesis during implantation.

After attachment and migration through the endometrial epithelium, the embryo must induce angiogenesis within the endometrial stroma to successfully complete the implantation process. Growth factors have been shown to play an important role in embryo implantation and placentation. The aim of the study was to investigate the expression of angiopoietin-1 and -2 (Ang-1 and -2) mRNA and protein expression during the development of single preimplantation mouse embryos and of possible complementary expression in mouse uteri. Angiopoietin-1 mRNA was expressed throughout development in 78% of zygotes, 66% of 2-cell-embryos, 71% of 4-cell-embryos, 70% of 8-cell-embryos, 60% of morula stages, 48% of early blastocysts and 78% of late blastocysts. The number of Ang-1-expressing embryos in the early-blastocyst group was significantly different in comparison with zygotes, 4-cell-embryos, 8-cell-embryos and late blastocysts. Angiopoietin-2 mRNA and protein expression could not be detected in preimplantation embryos. Examination of the uteri revealed Ang-2 mRNA and protein expression in the oestrogen-dominated cycling phase and the progesterone-dominated mated phase, whereas Ang-1 expression was restricted to the mated phase. Herein, Ang-1 expression in preimplantation mouse embryos as well as Ang-1 and -2 expression in mouse uteri is demonstrated, suggesting a possible role for angiopoietins in the embryo-maternal dialogue of the implantation process via an enhancement of the vascular remodelling in favour of an implanting conceptus.

Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women.

Histological evaluation of endometrium has been the gold standard for clinical diagnosis and management of women with endometrial disorders. However, several recent studies have questioned the accuracy and utility of such evaluation, mainly because of significant intra- and interobserver variations in histological interpretation. To examine the possibility that biochemical or molecular signatures of endometrium may prove to be more useful, we have investigated whole-genome molecular phenotyping (54,600 genes and expressed sequence tags) of this tissue sampled across the cycle in 28 normo-ovulatory women, using high-density oligonucleotide microarrays. Unsupervised principal component analysis of all samples revealed that samples self-cluster into four groups consistent with histological phenotypes of proliferative (PE), early-secretory (ESE), mid-secretory (MSE), and late-secretory (LSE) endometrium. Independent hierarchical clustering analysis revealed equivalent results, with two major dendrogram branches corresponding to PE/ESE and MSE/LSE and sub-branching into the four respective phases with heterogeneity among samples within each sub-branch. K-means clustering of genes revealed four major patterns of gene expression (high in PE, high in ESE, high in MSE, and high in LSE), and gene ontology analysis of these clusters demonstrated cycle-phase-specific biological processes and molecular functions. Six samples with ambiguous histology were identically assignable to a cycle phase by both principal component analysis and hierarchical clustering. Additionally, pairwise comparisons of relative gene expression across the cycle revealed genes/families that clearly distinguish the transitions of PE-->ESE, ESE-->MSE, and MSE-->LSE, including receptomes and signaling pathways. Select genes were validated by quantitative RT-PCR. Overall, the results demonstrate that endometrial samples obtained by two different sampling techniques (biopsy and curetting hysterectomy specimens) from subjects who are as normal as possible in a human study and including those with unknown histology, can be classified by their molecular signatures and correspond to known phases of the menstrual cycle with identical results using two independent analytical methods. Also, the results enable global identification of biological processes and molecular mechanisms that occur dynamically in the endometrium in the changing steroid hormone milieu across the menstrual cycle in normo-ovulatory women. The results underscore the potential of gene expression profiling for developing molecular diagnostics of endometrial normalcy and abnormalities and identifying molecular targets for therapeutic purposes in endometrial disorders.

Molecular cloning and characterization of a human adenocarcinoma/epithelial cell surface antigen complementary DNA.

A human adenocarcinoma-associated antigen (KSA) defined by the monoclonal antibody KS1/4 has become the focus of several site-directed strategies for tumor therapy. KSA, a 40,000 Da cell surface glycoprotein antigen, is found at a high density in all adenocarcinomas examined to date and in corresponding normal epithelial tissues. Here we describe the cloning and sequencing of overlapping complementary DNA clones which encode the entire KSA as expressed in UCLA-P3, a human lung adenocarcinoma cell line. We have deduced the 314-amino acid sequence and have compared it to the N-terminal amino acid sequence data of the affinity-purified antigen. The KSA is synthesized as a 314-residue-long preproprotein that is then processed to a 232-residue-long antigen. KSA appears to have a single transmembrane domain of 23 residues that separates the highly charged 26-residue cytoplasmic domain from the extracellular domain. The N-terminal region of the propeptide is rich in cysteines and contains three potential N-glycosylation sites. Computer-assisted analyses at both the DNA and protein levels have found no significant similarities of this protein to known sequences, but a GC-rich 5' terminus is evident. Northern blot analysis shows that transcription of KSA can be detected in RNA isolated from normal colon but not in RNA isolated from normal lung, prostate, or liver.

Retinoic acid inhibits junctional communication between animal cells.

Retinoic acid inhibits junctional communication between a variety of vertebrate cell types in culture. It reduces the intercellular transfer of 3H-nucleotides between Syrian hamster kidney fibroblasts (BHK 21/13), Chinese hamster lung fibroblasts (V79), rat liver epithelial cells (BRL), Swiss mouse embryo fibroblasts (3T3), rainbow trout gonadal fibroblasts (RTG2) and Xenopus embryo fibroblasts (Xen). It also reduces metabolic cooperation between hypoxanthine-guanine phosphoribosyl transferase deficient mutant and wild-type BHK cells. The inhibition is rapid (intercellular transfer of iontophoretically injected Lucifer Yellow CH between BRL cells is completely blocked after the cells have been exposed to 10(-4) M retinoic acid for 5 min), and is fully reversed when the drug is removed. Based on these results and the observation that the amount of gap junctional protein isolated from cells grown in the presence of retinoic acid for 1 h is the same and after 24 h is increased (1.3- to 3.1-fold) compared with the amount isolated from untreated cells, we suggest that the inhibitory effect is mediated by the reversible closure of junctional channels.

A method for combined C-banding and silver staining.

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.

Analysis of vertebrate gap junction protein.

A new method for the purification of gap junctions is described which depends on the extraction of cell monolayers or tissue homogenates with Triton X-100. The major band on SDS-polyacrylamide gel electrophoresis (PAGE) of junctional preparations from a variety of vertebrate sources has an apparent mol. wt. of 16,000 (16 K). Further evidence for the junctional origin of the 16 K protein is provided by the results of four different experimental approaches. (i) The junctions form a sharp band in potassium iodide density gradients at 1.195 g/cm3 and the 16 K protein is the only detectable band in fractions of this bouyant density. (ii) The junctions are progressively solubilised by increasing concentrations of SDS (in the range 0.1-0.5%) and the dissolution of the junctional structure, observed by electron microscopy, parallels the release of the 16 K protein. (iii) Glutaraldehyde fixation of intact junctions cross-links the 16 K protein. (iv) The recoverable amount of the 16 K protein correlates with known changes in gap junctional area in the regenerating weanling rat liver after partial hepatectomy and in V79 cell cultures exposed to 4beta-phorbol 12-myristate 13-acetate.