ELK Foundation for Health Research

Crieff, United Kingdom

ELK Foundation for Health Research

Crieff, United Kingdom
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Iles R.K.,St. Bartholomew's Hospital | Iles R.K.,ELK Foundation for Health Research | Iles R.K.,MAP Diagnostics Ltd. | Cole L.A.,United States hCG Reference Service | And 2 more authors.
International Journal of Molecular Sciences | Year: 2014

The analysis of human chorionic gonadotropin (hCG) in clinical chemistry laboratories by specific immunoassay is well established. However, changes in glycosylation are not as easily assayed and yet alterations in hCG glycosylation is associated with abnormal pregnancy. hCGβ-core fragment (hCGβcf) was isolated from the urine of women, pregnant with normal, molar and hyperemesis gravidarum pregnancies. Each sample was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) analysis following dithiothreitol (DTT) reduction and fingerprint spectra of peptide hCGβ 6-40 were analyzed. Samples were variably glycosylated, where most structures were small, core and largely mono-antennary. Larger single bi-antennary and mixtures of larger mono-antennary and bi-antennary moieties were also observed in some samples. Larger glycoforms were more abundant in the abnormal pregnancies and tri-antennary carbohydrate moieties were only observed in the samples from molar and hyperemesis gravidarum pregnancies. Given that such spectral profiling differences may be characteristic, development of small sample preparation for mass spectral analysis of hCG may lead to a simpler and faster approach to glycostructural analysis and potentially a novel clinical diagnostic test. © 2014 by the authors; licensee MDPI, Basel, Switzerland.

Butler S.A.,Middlesex University | Luttoo J.,Middlesex University | Freire M.O.T.,Whittington Hospital | Abban T.K.,Middlesex University | And 2 more authors.
Reproductive Sciences | Year: 2013

Human chorionic gonadotropin (hCG) is produced by trophoblast cells throughout pregnancy, and gene expression studies have indicated that hCG-beta subunit (hCGβ) expression is active at the 2 blastomere stage. Here, we investigated the qualitative hCG output of developing embryos in culture and hCG isoforms expressed in the secretome as a novel sensitive method for detecting hCG. Culture media was collected from the culture plates of 118 embryos in culture (including controls and embryos at different stages of culture) from 16 patients undergoing routine fertility treatment. The hCGβ was detectable in media from 2 pronuclear (2PN) stage embryos through to the blastocyst stage. The hCGβ was absent in 1PN and arrested embryos as well as all media controls. Prior to hatching, hyperglycosylated hCG (hCGh) was observed selectively in 3PN embryos, but after hatching, along with hCG, became the dominant hCG molecule observed. We have reported at the 2PN stage the earliest evidence of hCGβ expression in embryos. There is a suggestion this may be indicative of quality in early embryos, and hCGh seen at the pronuclear stage may suggest triploid abnormality. The dominance of hCG, and hCGh expression, seen after blastocyst hatching may be indicative of potential implantation success. Thus, hCG isoforms have potential roles as biomarkers of embryo viability for embryo/blastocyst transfer. © The Author(s) 2013.

Burczynska B.B.,Middlesex University | Burczynska B.B.,Poznan University of Medical Sciences | Kobrouly L.,Middlesex University | Butler S.A.,Middlesex University | And 5 more authors.
Anticancer Research | Year: 2014

Background: Ectopic secretion of human chorionic gonadotrophin free beta (hCGβ0) by epithelial cancer is associated with aggressive tumors which more readily metastasize, possibly by acting as an autocrine anti-Apoptotic agent. hCGβ0 is encoded by six homologous CGB genes, with poorly-understood variable transcriptionally active expression profiles; CGB1 and CGB2 have always been considered pseudogenes. However, transcripts from CGB1 and-2 can be detected in placental, testicular and pituitary tissues. The expression and function of these genes in cancer is less wellknown. Materials and Methods: Expression profiles of CGB genes in epithelial cancer cells by quantitative polymerase chain reaction (qPCR) were explored, along with the consequence of specific siRNA silencing of CGB1 and 2. Immunohistochemical and immunoassay techniques were used to detect the translation and secretion of hCGβ0 in these cells. Results: CGB1 and-2\par gene transcripts were only detected in cells which secreted hCGβ0. siRNA-mediated silencing of CGB1 and-2 transcripts significantly reduced secreted protein in concordance with a reduction in cell survival to a greater degree than that of other CGB genes. Conclusion: CGB genes 1 and 2, previously considered as pseudogenes, are notably expressed by epithelial cancer cell lines. The transcription of these genes, but not other CGB genes, correlates with a functionally expressed protein and propensity for cancer growth.

Burczynska B.,Middlesex University | Burczynska B.,Poznan University of Medical Sciences | Booth M.J.,London Metropolitan University | Iles R.K.,Middlesex University | And 6 more authors.
Anticancer Research | Year: 2013

Background: Expression of human chorionic gonadotropin beta subunit (hCGβ) by epithelial carcinomas is associated with a poor prognosis and has a proposed autocrine growth effect on cancer cells by inhibition of apoptosis. Material and Methods: We transduced the hCGβ-expressing bladder cancer cell line SCaBER with short hairpin (sh) RNA lentiviral gene-specific (CGB) constructs and determined its impact on the synthesis of hCGβ and the resultant effect on cancer cell growth. Results: Stable CGB gene-silenced clones exhibited a 60%-80% reduction in the level of hCGβ expressed and a reduced growth rate of more than 40% compared to wild-type SCaBER cells. Conclusions: shRNA Lentiviral particles achieve stable knockdown of hCGβ translation in the bladder cancer cell line SCaBER. This transforms the phenotype by reducing hCGβ expression and cell growth rate. This is consistent with the proposed autocrine/paracrine function of ectopic hCGβ expression during oncogenesis.

Butler S.A.,Middlesex University | Abban T.K.A.,Middlesex University | Borrelli P.T.A.,Bedford Hospital NHS Trust | Luttoo J.M.,Middlesex University | And 2 more authors.
Clinical Biochemistry | Year: 2013

Objectives: Tubal rupture as a result of an ectopic pregnancy is the leading cause of first trimester maternal mortality. Currently, the diagnosis of ectopic pregnancy depends on transvaginal ultrasound and serial serum measurements of human chorionic gonadotrophin (hCG), which requires follow up. The objective of this study was to examine whether single point measurements at presentation could distinguish between women with ectopic pregnancy, viable pregnancy, and spontaneous miscarriage. Design and methods: Serum total hCG (hCGt), hyperglycosylated hCG (hCGh), free beta subunit of hCG (hCGβ), progesterone (P), and CA-125 were measured by chemiluminescence immunoassay over a 3. month period in 441 women presenting at the emergency room with abdominal pain and a positive pregnancy test. Patient outcomes were followed and confirmed by histology. 65 samples were excluded due to poor sample storage, or lost to follow up. Results: The pregnancy outcomes were 175 viable pregnancies, 175 spontaneous miscarriages, and 26 ectopic pregnancies. A serum hCGt <. 3736. mIU/mL cut off was 100% sensitive, with 76% specificity, for distinguishing ectopic pregnancy from viable pregnancy; but did not differentiate spontaneous miscarriage. Serum CA125 <. 41.98. U/mL produced 100% sensitivity and 43% specificity in distinguishing ectopic pregnancy from spontaneous miscarriage. Sequential application of hCGt and CA-125 cut off followed by ultrasound could detect 100% of ectopic pregnancies with 87% specificity for all intrauterine pregnancies. Conclusion: The combination of serum hCGt <. 3736. mIU/mL, followed by CA125 <. 41.98. U/mL is a promising algorithm for detecting all ectopic pregnancy at initial presentation. © 2013 The Canadian Society of Clinical Chemists.

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