Rectal suction biopsies to diagnose Hirschsprung's disease in a low-resource environment - optimising cost-effectiveness.

BACKGROUND: The diagnosis of Hirschsprung's disease (HD) by rectal suction biopsy (RSB) has cost implications that could be reduced by ascertaining the optimal number of specimens required. The aim was to audit our experience to optimise cost-effectiveness. METHODS: Medical records of all patients who underwent an RSB between January 2018 and December 2021 were reviewed. In 2020, we transitioned from using the Solo-RBT to the rbi2 system (requiring single-use cartridges). Descriptive statistics were reported and a comparative analysis of the diagnostic efficacy of the Solo-RBT versus the rbi2 system was performed. The cost of consumables was calculated according to the number of specimens submitted. RESULTS: Of 218 RSBs, 181 were first and 37 were repeat. The mean age at biopsy was 62 days (IQR 22-65). An average of two tissue specimens were obtained per biopsy. Of the 181 first biopsies, 151 were optimal and 30 suboptimal. HD was confirmed in 19 (10.5%) of the patients. Amongst biopsies where a single specimen was obtained, 16% were inconclusive, compared to 14% with two specimens and 5% with three specimens. The cartridges for the rbi2 system cost R530. If two cartridges are used at initial biopsy the total cost is double of a single tissue specimen sent for initial biopsy, and two specimens sent for repeat biopsies. CONCLUSION: In a low-resource setting, selecting the appropriate RSB system and obtaining a single specimen is sufficient to diagnose HD. Patients with inconclusive results should undergo a repeat biopsy where two specimens are obtained.

The possible biological and reproductive functions of ubiquitin.

The protein ubiquitin (Ub) appears to be present in all eukaryotic cells. Its widespread presence and extremely conserved structure indicate that it may play a vital role in cell metabolism. The roles of Ub are mediated by its covalent attachment to target proteins, a process known as ubiquitylation, a form of protein modification which may lead to degradation of the modified protein. A number of proteins with similar structure to Ub but varying in function have been isolated. Recently, there has been much interest in the role of Ub and its related proteins in reproductive processes. Ub and Ub-related proteins may be involved in gametogenesis, modulation of steroid receptor concentrations, placental development and endometrial modification at the beginning of pregnancy. These wide-ranging effects have led to extensive research which will be reviewed in this article.

Lectin binding characteristics of mouse placental cells.

The lectin binding characteristics of mouse placental cells were examined. Wax embedded tissue sections of placentae from d 14 pregnant mice were stained with 26 lectins, with a wide range of sugar specificities. Cell cultures prepared from d 14 mouse placentae and cultured for 24 h were stained with 7 of the lectins to determine if they could be used as markers for the different trophoblast cells in culture. In tissue sections all placental cell populations bound lectin but no lectin bound specifically to any single trophoblast population. All the lectins which bound to layer 1 cytotrophoblast lining the maternal blood spaces of the labyrinthine placenta also bound to the fetal endothelium of the labyrinthine placenta. Binding of lectin appeared strongest on the adluminal membrane of these cell populations suggesting a role for the carbohydrate moieties in nutrient transfer. Few lectins bound to junctional zone trophoblast. Overall, the binding of lectin to cultured cells did not correlate exactly with lectin binding to the cell populations in tissue sections. The value of lectins as markers for placental cells in culture was therefore found to be limited. Our findings indicate that carbohydrate expression by at least some placental cells may vary in culture from that expressed by the cells in vivo with obvious concerns for the validity of functional in vitro studies.

The progesterone receptor and ubiquitin are differentially regulated within the endometrial glands of the natural and stimulated cycle.

The initiation of human pregnancy requires precisely timed development of the endometrium to receive the implanting blastocyst. The ovarian steroid hormones are essential for development and maintenance of a hospitable uterine environment. The hormonal regimes employed in assisted reproduction procedures are known to alter the abundance of specific endometrial receptors for these steroids. Since, in the presence of ligand, the progesterone receptor (PR) is known to be modified by the small intracellular protein ubiquitin, we have investigated the localization of ubiquitin and PR within the endometrial glands of 28 fertile women during a monitored menstrual cycle and also during a stimulated cycle prior to oocyte donation. We have also observed the number of gland cells undergoing cell division as demonstrated by the presence of Ki67 immunostaining. We demonstrate that the percentage of ubiquitin-positive nuclei increases from day four post-ovulation to day 10 post-ovulation in the natural cycle, but that this increase is not seen during a stimulated cycle. The presence of PR within glandular epithelium and the proliferation of gland cells were only observed during the early secretory phase and did not appear to vary significantly between the two cycles. We conclude that ubiquitin may play an important role in endometrial development and that perturbation of ubiquitin may be related to the lower implantation rate seen in the stimulated cycle.

Ubiquitin and ubiquitin-protein conjugates are present in human cytotrophoblast throughout gestation.

Ubiquitin is a small protein involved in many intracellular processes. We have previously shown that levels of ubiquitin change during the process of decidualisation in the human uterus at the beginning of pregnancy. Other workers have shown that the ubiquitin system may be essential for normal murine placental development. In this investigation we employed immunohistochemistry and immunoblotting techniques to study the distribution and abundance of ubiquitin and ubiquitin-protein conjugates within human placental specimens from throughout gestation. Trophoblast from two pathological conditions, ectopic pregnancy and pregnancy-induced hypertension (PIH), was also investigated. Ubiquitin was detected within both the cytoplasm and nucleus of the cytotrophoblast layer only. Both monomeric and conjugated forms of ubiquitin were detected. The relative abundance of ubiquitin did not change through gestation or in the two disorders of pregnancy studied. Ubiquitin cross-reactive protein was not detected in the tissues of interest. This is the first report to demonstrate the cell-specific localisation of ubiquitin and ubiquitin-protein conjugates in the human cytotrophoblast and provides supportive evidence that ubiquitin may be important during placental development.

Ubiquitin cross-reactive protein gene expression is increased in decidualized endometrial stromal cells at the initiation of pregnancy.

Ubiquitin cross-reactive protein (UCRP; also known as ISG15) is an interferon-upregulated protein implicated in the response to viral infection. An equivalent protein has been demonstrated within the bovine uterus in early pregnancy in response to conceptus-derived interferon-tau. We have previously shown the upregulation of UCRP within decidualized stromal cells of the human and non-human primate endometria. We now show that an increase in UCRP gene expression within the decidualized stromal cell accompanies these increased protein concentrations. Using Northern blotting techniques we demonstrate basal concentrations of URCP mRNA within the non-pregnant endometrium and an increase in signal within some decidual specimens of first trimester decidua. This signal may represent increased URCP transcription within the sub-population of stromal cells that undergo decidualization, since this cell type exhibits an increase in UCRP message as detected by in-situ hybridization.

Localization of ubiquitin and ubiquitin cross-reactive protein in human and baboon endometrium and decidua during the menstrual cycle and early pregnancy.

We have examined the distribution of ubiquitin and the related ubiquitin cross-reactive protein (UCRP) in paraffin-embedded sections of human and baboon endometrium and decidua by immunoperoxidase or immunofluorescence cytochemistry with antibodies raised against ubiquitin, UCRP, CD45, and insulin-like growth factor-binding protein-1. Anti-ubiquitin immunoreactivity was present in the nonpregnant endometrium, particularly in the glandular epithelial cells, and up-regulated in endometrial stromal cells as they decidualized at the beginning of pregnancy. Anti-UCRP immunoreactivity was absent from nonpregnant tissue but accumulated to high levels in decidual cells during pregnancy. Western blotting indicated that immunoreactivity was primarily due to the presence of ubiquitin and UCRP conjugated to other proteins, and that although levels of ubiquitin-protein conjugates do not change substantially during pregnancy, decidualization is accompanied by the appearance of conjugates of UCRP. Baboon uterine tissues demonstrated a similar distribution of the two proteins, which indicates that the baboon may be a useful model for study of the role of the ubiquitin system and UCRP in the establishment of pregnancy in humans.