Josephine A. Drury

Comparison of urinary and plasma malondialdehyde in preterm infants

The measurement of malondialdehyde (MDA) in biological fluids remains a popular method for the quantification of free radical damage to lipids in vivo. Several diseases of prematurity are thought to be related to oxidative injury and previous studies have found elevated MDA in plasma and urine in preterm infants. Our aim was to investigate the relationship between plasma and urinary MDA levels in preterm infants during the first week of life using a high-performance liquid chromatography (HPLC) based, thiobarbituric acid (TBA) assay with paired plasma and urine samples. We obtained 50 paired samples, and were unable to demonstrate a relationship between the two parameters after the first day of life. In 18 cases a further urine sample was collected 24 h later. There was a positive correlation (r = 0.54, P = 0.02) between plasma MDA and urinary MDA 24 h later. The finding that plasma changes in MDA are reflected in urine 24 h later validates the use of urinary MDA as a marker of whole body lipid peroxidation in populations without renal disease.

Endometriosis is associated with aberrant endometrial expression of telomerase and increased telomere length

BACKGROUND In order to test our hypothesis that endometriosis is associated with abnormal expression of telomerase and telomere lengthening in endometrium, we assessed endometrial expression of the human telomerase enzyme and telomere length (TL). METHODS This prospective pilot study, included 29 women with symptomatic, surgically diagnosed endometriosis (Group 1) and 27 healthy, fertile, symptom-free women without endometriosis (Group 2, confirmed by laparoscopy). Seventeen women in Group 1 and 15 women in Group 2 had endometrial biopsies taken on Day 21 +/- 2 of the cycle. A further 12 women in each group were biopsied on Day 26 +/- 2. Telomerase and estrogen receptor beta (ERbeta) expression was evaluated by immunohistochemistry. Mean TL was determined by quantitative PCR. RESULTS The endometria of fertile healthy women showed either weak or no telomerase immunoreactivity throughout the luteal phase. Immunostaining for telomerase was significantly increased during the implantation window and the premenstrual endometria of women with endometriosis (P < 0.0001). This was associated with a loss of stromal and vascular ERbeta immunostaining (P < 0.05). The mean TL were significantly longer in endometria of women with endometriosis during the implantation window (P = 0.005), indicating the biological relevance of our novel finding of telomerase in benign endometrium. There was positive correlation of the circulating estradiol with peripheral blood TL in women. CONCLUSIONS We speculate that aberrant endometrial expression of telomerase mediates alterations in cell fate that enhance proliferation, contributing to the pathogenesis of endometriosis.

Breath pentane as a marker for lipid peroxidation and adverse outcome in preterm infants

AIM To test the hypothesis that complications of neonatal intensive care are related to increased oxygen derived free radical activity, using breath pentane as a marker of lipid peroxidation. METHODS Exhaled breath was collected daily from 57 ventilated preterm infants and pentane concentration measured by gas chromatography. RESULTS High peak pentane exhalation was significantly associated with low gestational age, mortality, intraventricular haemorrhage and retinopathy of prematurity. Peak pentane was not significantly associated with the development of chronic lung disease. CONCLUSIONS The demonstration that pentane exhalation is related to the course of neonatal disease and its outcome is consistent with the hypothesis that lipid peroxidation is associated with these illnesses, and may contribute to their severity. If this is a causal relation, antioxidant treatments could prove useful in reducing their severity. Measurement of breath pentane might assist in the assessment of antioxidant strategies prior to more extensive clinical trials.

Localization of angiogenic growth factors and their receptors in the human endometrium throughout the menstrual cycle and in recurrent miscarriage

BACKGROUND Angiogenesis is a key feature of endometrial development. Inappropriate endometrial vascular development has been associated with recurrent miscarriage (RM) with increased amounts of perivascular smooth muscle cells surrounding them. METHODS In the current study, we have used immunohistochemistry to study temporal and spatial expression of a series of angiogenic growth factors (AGFs) and their receptors; vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF-D, VEGF-R1, VEGF-R2, VEGF-R3, platelet-derived growth factor (PDGF)-BB, PDGF-Rα, PDGF-Rβ, transforming growth factor (TGF)-β1, TGF-βRI, TGF-βRII, angiopoietin (Ang)-1, Ang-2 and Tie-2, in the proliferative, early secretory and mid-late secretory phase endometrium from control women as well as in the mid-late secretory phase of women with a history of RM. The AGFs and their receptors studied were immunostained and assessed separately in stromal, vascular smooth muscle, endothelial and glandular epithelial cells. Laser capture microdissection and real-time RT-PCR were used to confirm expression patterns observed by immunohistochemistry. RESULTS Most AGFs investigated showed both temporal and spatial expression patterns in normal cycling endometrium. In addition, immunostaining intensity for several AGFs was altered in women with a history of RM, particularly in vascular smooth muscle cells (VSMCs). VSMC expression of TGF-β1, VEGF-R1 and VEGF-R2 was increased while expression of PDGF-BB, TGF-βRI, TGF-βRII, Ang-2, VEGF-A and VEGF-C was reduced. CONCLUSIONS This study confirms that the cycling endometrium is a highly angiogenic tissue and that this process is likely to be altered in women with a history of RM and may contribute to the aetiology of this condition.

SSEA-1 isolates human endometrial basal glandular epithelial cells: phenotypic and functional characterization and implications in the pathogenesis of endometriosis

STUDY QUESTION Can the basal epithelial compartment of the human endometrium be defined by specific markers? SUMMARY ANSWER Human endometrial epithelial cells from the basalis express nuclear SOX9 and the cell-surface marker SSEA-1, with some cells expressing nuclear β-catenin. In vitro, primary endometrial epithelial cells enriched for SSEA-1+ show some features expected of the basalis epithelium. WHAT IS KNOWN ALREADY The endometrial glands of the functionalis regenerate from the basalis gland stumps following menstruation. Endometriosis is thought to originate from abnormal dislocation of the basalis endometrium. In the highly regenerative intestinal epithelium, SOX9 and nuclear β-catenin are more highly expressed in the intestinal crypt, the stem/progenitor cell region. STUDY DESIGN, SIZE, DURATION A large prospective observational study analysing full-thickness human endometrial hysterectomy samples from 115 premenopausal women, 15 post-menopausal women and ectopic endometriotic lesions from 20 women with endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS Full-thickness endometrium from hysterectomy tissues was analysed by immunohistochemistry for SSEA-1, SOX9 and β-catenin. Primary human endometrial epithelial cells from short-term cultures were sorted into SSEA1+/- fractions with a cell sorter or magnetic beads and analysed for markers of differentiation and pluripotency and telomere lengths (TLs) using qPCR, telomerase activity [telomere repeat amplification protocol (TRAP)] and growth in 3D culture. MAIN RESULTS AND THE ROLE OF CHANCE Similar to the intestinal crypt epithelium, human endometrial basal glandular epithelial cells expressed nuclear SOX9 and contained a rare subpopulation of cells with nuclear β-catenin suggestive of an activated Wnt pathway. The embryonic stem cell-surface marker, SSEA-1, also marked the human endometrial basal glandular epithelial cells, and isolated SSEA-1(+) epithelial cells grown in monolayer showed significantly higher expression of telomerase activity, longer mean TLs, lower expression of genes for steroid receptors and produced a significantly higher number of endometrial gland-like spheroids in 3D culture compared with SSEA-1(-) epithelial cells (P = 0.009). Cells in ectopic endometriosis lesions also expressed SSEA-1 and nuclear SOX9, suggesting that the basalis contributes to ectopic lesion formation in endometriosis following retrograde menstruation. LIMITATIONS, REASONS FOR CAUTION This is a descriptive study with only short-term culture of the primary human epithelial cells in vitro. WIDER IMPLICATIONS OF THE FINDINGS The surface marker SSEA1 enriches for an endometrial epithelial cell subpopulation from the basalis. Since the functional endometrium originates from these cells, it is now possible to study basalis epithelium for stem/progenitor cell activity to extend our current understanding of endometrial biology in health and diseases. STUDY FUNDING/COMPETING INTEREST(S) The work included in this manuscript was funded by Wellbeing of Women project grant RG1073 (D.K.H. and C.G.). We also acknowledge the support by National Health and Medical Research Council, RD Wright Career Development Award 465121 and Senior Research Fellowship 1042298, and the Victorian Governments Operation Infrastructure Support Program to C.G. and MRC G0601333 to T.V.Z. All authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER N/A.

PENTANE MEASUREMENT IN VENTILATED INFANTS USING A COMMERCIALLY AVAILABLE SYSTEM

The measurement of breath pentane as a marker of lipid peroxidation has recently been criticized. Problems encountered include the coelution of isoprene with pentane, contamination with exogenous pentane, and the influence of elevated oxygen concentration. The aim of this project was to investigate and evaluate the Chrompack 9001 Gas Chromatograph, using thermal desorption and cryofocussing and an Al2O3/KCl PLOT column with FID, for use in the measurement of breath pentane in ventilated preterm infants. We have clearly separated isoprene from n-pentane and used hydrocarbon free air to clear the airways and avoid contamination with exogenous pentane. Samples should be stored in Tedlar bags for a maximum of 48 h and on capped desorption tubes for no longer than 24 h. Patient variability was relatively high (mean 18%, n = 4); thus, all patients were sampled in duplicate. No correlation was found between fractional inspired oxygen concentration (FiO2) and exhaled pentane in preterm infants ventilated for respiratory distress syndrome. In conclusion, we feel that despite the pitfalls and technical difficulties, with careful attention to detail it is possible to reliably measure breath pentane in ventilated preterm infants as an index of lipid peroxidation.

Sustained replication in endometrium of women with endometriosis occurs without evoking a DNA damage response

BACKGROUND To test our hypothesis that eutopic secretory phase endometrium from women with endometriosis is similar to proliferative phase endometrium from fertile women without endometriosis, we explored the expression of regulators of cell fate across the menstrual cycle. METHODS Endometrial biopsies were taken from 73 women, comprising 38 women with surgically diagnosed active peritoneal endometriosis (Group 1) and 35 fertile women without endometriosis (Group 2). Nucleolin, proliferating cell nuclear antigen (PCNA), telomerase and histone gamma-H2AX expression was evaluated by immunohistochemistry and mean telomere length (TL) by quantitative PCR. RESULTS We have immunolocalized nucleolin and gamma-H2AX in the benign premenopausal endometrium for the first time. All markers were present in the proliferative phase endometrium of all women. In Group 2, during the secretory phase, proliferative markers declined with a paradoxical increase in stromal gamma-H2AX. Women in Group 1, however, showed a persistent immunoreactivity for the proliferative markers, while the staining for gamma-H2AX decreased in secretory endometrium (P < 0.05). This difference between groups was significant in both stroma and glands for nucleolin (P < 0.0001), PCNA (P < 0.01) and gamma-H2AX (P < 0.05) in the secretory phase. We showed a positive correlation between mean TL and nucleolin expression (glandular r = 0.37, P = 0.002; stromal r = 0.4, P = 0.001), telomerase immunoreactivity (glandular r = 0.33, P = 0.009; stromal r = 0.4, P = 0.001) and glandular PCNA (r = 0.35, P = 0.004), whereas a negative correlation was seen between mean TL and gamma-H2AX (r = -0.28, P = 0.04). CONCLUSIONS These findings demonstrate that the state of replication seen in secretory phase endometrium from women with active peritoneal endometriosis is not a simple extension of the proliferative phase.

Aberrant expression of regulators of cell-fate found in eutopic endometrium is found in matched ectopic endometrium among women and in a baboon model of endometriosis

BACKGROUND We have recently shown that women with endometriosis express an increased amount of telomerase and nucleolin, with concomitant loss of γ-H2AX in eutopic endometrium. To further examine these selected factors that regulate cell fate, in the pathogenesis of endometriosis, we studied the expression of telomerase, nucleolin, proliferating cell nuclear antigen and γ-H2AX in ectopic endometriotic deposits from women, and in matched eutopic and ectopic endometrial tissue from a baboon model of endometriosis. METHODS Ectopic active peritoneal endometriotic lesions were collected from seven symptomatic women. Endometriosis was induced in six baboons by intra-peritoneal autologous inoculation of menstrual endometrium. Eutopic and matched ectopic endometrial tissues were collected prior to and 6, 12 and 15 months after the induction of endometriosis as previously described. Eutopic endometrium was also obtained from eight healthy fertile control baboons. Immunohistochemistry was performed as previously described, and telomerase activity was confirmed using the telomeric repeat amplification protocol assay. RESULTS All active human endometriotic lesions expressed the proliferative markers but showed weak or absent staining for γ-H2AX. A similar expression pattern of these markers was seen in the ectopic lesions of the baboons with induced disease. In these baboons, the eutopic endometrium also showed intense immunoreactivity for all proliferative markers 6-12 months after induction with a parallel loss of γ-H2AX. The opposite staining pattern was seen in eutopic endometrium of healthy animals and in pre-induction endometrium of animals with induced disease. CONCLUSIONS Endometriotic lesions have excess proliferative potential; in baboons, these were present within 12 months of the initiation of the disease. In eutopic tissue, these changes appear to be induced by the development of endometriosis.

Endometrial cell counts in recurrent miscarriage: a comparison of counting methods

Drury J A, Nik H, van Oppenraaij R H F, Tang A‐W, Turner M A & Quenby S 
(2011 Histopathology 59, 1156–1162 
Endometrial cell counts in recurrent miscarriage: a comparison of counting methods

Reduction of oxidative stress marker in lung fluid of preterm infants after administration of intra-tracheal liposomal glutathione

Background: Low levels of glutathione are associated with subsequent chronic lung disease in preterm infants. Incorporation of glutathione into liposomes offers a method of increasing levels with a prolonged half-life compared with direct inhalation. Objectives: The aim of this study was to examine the clinical feasibility of administering a single dose of liposomal glutathione and its effectiveness at raising glutathione at 12 and 24 h after treatment. Methods: Fourteen ventilated preterm infants from the Regional Neonatal Intensive Care Unit at Liverpool Women’s Hospital received 1 mg/kg or 10 mg/kg liposomal glutathione intra-tracheally and bronchoalveolar lavage fluid was collected prior to treatment, 12 and 24 h after dosing for glutathione and malondialdehyde estimation. Results: Mean glutathione was initially 12.2 µmol/l, increasing to 52.8 µmol/l at 12 h (p = 0.006). Mean malondialdehyde was initially 265.6 nmol/l decreasing to 11.2 nmol/l at 12 h (p = 0.018). Conclusions: Intra-tracheal liposomal glutathione instillation offers a feasible method of raising pulmonary glutathione in preterm infants and shows biochemical antioxidant effects.