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STUDY QUESTION: Can bioinformatics analysis of publically available microarray datasets be utilized in identifying potentially important transcription factors (TF) in the hormonal regulation of the endometrium? SUMMARY ANSWER: Systems integration and analysis of existing complex (published) datasets, predicted a role for the novel transcription factor, Forkhead Box D3 (FOXD3) in healthy endometrium and in endometriosis, which was followed by the demonstration of decreased levels of the protein upon decidualisation of normal human endometrial stromal cells in vitro and differential endometrial expression in the stroma in endometriosis. WHAT IS KNOWN ALREADY: The reported endometriosis-associated endometrial aberrations are most pronounced in the progesterone-dominant secretory phase and progesterone resistance is a proposed causative factor. STUDY DESIGN, SIZE, DURATION: The study was initially an 'in silico' study, with confirmatory 'wet lab' data from western blotting (WB), qPCR and Immunohistochemistry (IHC) on endometrial biopsies obtained from 142 women undergoing gynaecological surgery. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was conducted at a University Research Institute. Bioinformatic analysis of selected published microarray datasets identified differentially regulated genes for the early and mid-secretory phases relative to the proliferative phase. Diseases and Functions categories were identified with Ingenuity (IPA) 'core analysis' software. The key transcription factors controlling secretory phase gene changes were revealed with oPOSSUM software. FOXD3 expression levels were examined in human endometrial samples from women aged 18-55 years by WB, IHC, and qPCR. The progesterone regulation of endometrial FOXD3 levels was examined in vivo and in cultured primary human endometrial stromal cells in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: Initial data mining and subsequent bioinformatics analysis of human endometrial microarray datasets identified FOXD3 to be a key regulator of gene expression specific to secretory phase/endometriosis. FOXD3 was dynamically expressed in healthy endometrium and differentially expressed in endometriosis. In vitro decidualisation of primary endometrial stromal cells significantly decreased FOXD3 protein (P = 0.0005) and progestagen (Levonorgestrel) treatment also reduced the high endometrial FOXD3 protein (P = 0.0001) and mRNA levels (P = 0.04) seen in untreated women with endometriosis, with a shift of FOXD3 from the nucleus to the cytoplasm. LIMITATIONS, REASONS FOR CAUTION: The quality of Bioinformatics analysis and results depends on the published micro-array data. WIDER IMPLICATIONS OF THE FINDINGS: An in depth analysis of FOXD3 function and its relationship with estrogen and progesterone might provide insights into its potential deregulation in proliferative disorders of the endometrium including endometrial cancer where its expression is also deregulated. Further, FOX transcription factors are increasingly seen as novel therapeutic targets in disease. STUDY FUNDING/COMPETING INTERESTS: We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H., A.J.V.). We also acknowledge the support of Liverpool Women's Hospital Foundation Trust (J.A.D.), Institute of Translational Medicine (D.M., A.J.V., D.K.H.) and the Institute of Integrative Biology (O.V.), University of Liverpool. All authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

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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.

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In order to assess whether markers of cell senescence are related to reproductive failure, the expression of telomerase and telomere length in endometrial biopsies from women with and without reproductive failure were assessed. This pilot study included 45 women of whom 10 had idiopathic recurrent loss of empty gestational sacs, 10 had idiopathic recurrent fetal loss (miscarriage following identification of fetal cardiac activity), 10 had recurrent implantation failure and 15 had two or more normal pregnancies (control group). An endometrial sample was collected during the window of implantation from each woman. The mean endometrial telomere length was determined by quantitative polymerase chain reaction. Telomerase expression was evaluated by immunohistochemistry. The endometria of the control group showed virtually no telomerase immunoreactivity during the window of implantation. However, the immunostaining for telomerase was significantly and differentially increased in various endometrial cellular compartments in women with recurrent reproductive failure (P < 0.05). There were no significant differences in mean telomere length between groups. These data provide a novel insight into the biological correlates of clinical types of recurrent reproductive failure and suggest that specific alterations in the regulation of endometrial cell fate are associated with different types of recurrent reproductive failure.

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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.

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AIMS: Stage 3 retinopathy of prematurity (ROP) usually develops in the very small babies. An infant born at 30 (2) weeks' gestation and of birth weight 2102 g was found to have stage 3 ROP bilaterally. The unusual severity of the disease in a child of birth weight >2000 g prompted further investigation. The aim was to look for any genetic causes for ROP. METHODS: Chromosomal analysis followed by vasculoendothelial growth factor (VEGF) analysis. RESULTS: Unbalanced translocation 18p (monosomy) and 6p (trisomy). Overexpression of VEGF. CONCLUSIONS: Overexpression of VEGF contributed to the unusual severity of ROP in this relatively large baby.

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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.

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Several diseases of prematurity are thought to be related to oxidative injury and many of the available markers are unsatisfactory. An assay was developed using HPLC with electrochemical detection for the quantitation of urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a proposed indicator for oxygen-derived free radical injury to DNA in preterm infants. A median value of 3.79 micromol/mol creatinine was obtained for normal children (2-15 years old, n=14). Urinary 8-OHdG excretion in neonates ranged from 0-99 micromol/mol creatinine. There were no gestation or birthweight related differences in urinary 8-OHdG, and no correlation with urinary malondialdehyde. Mean 8-OHdG excretion increased with postnatal age (r=0.80, p < 0.0001, n=15), mirroring the growth velocity curve. These changes could also be due to changes in the activity of the enzyme responsible for 8-OHdG excision. Urinary 8-OHdG levels are unlikely to accurately reflect oxygen derived free radical activity given the strength of the relationship with growth.

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1. In healthy humans, a balance exists between oxygen-derived free-radical production and their removal by antioxidants. In preterm infants inadequate antioxidant defences may contribute to the pathogenesis of some of the complications of prematurity. 2. Plasma total antioxidant status and malondialdehyde concentration were measured during the first 11 days of life in 25 infants to determine whether increased lipid peroxidation is associated with low extracellular antioxidant status. In a second group of infants, total antioxidant status was quantified within 12 h of birth, and subsequently on days 4 and 10 to investigate the hypothesis that adverse neonatal outcome is associated with low antioxidant status. 3. There may be a weak negative correlation between the total antioxidant status of infants and the lipid peroxidation marker malondialdehyde in plasma (r = -0.24, P = 0.056, n = 89) during the first 11 days of life. In the second group of infants, total antioxidant status was found to be significantly related to plasma urate and bilirubin levels, but not to adverse neonatal outcomes such as chronic lung disease, intraventricular haemorrhage, retinopathy of prematurity or death. 4. If adverse neonatal outcomes are due to inadequate antioxidant defences, these are likely to be intracellular or localized rather than general extracellular deficiencies.

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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.

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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.

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UNLABELLED: Frequent blood transfusions may produce changes in iron status which can give rise to oxygen-derived free-radical (ODFR) generation and oxidative injury. Preterm infants developing chronic lung disease (CLD) receive significantly more transfusions. A total of 73 very preterm infants had weekly estimations of serum iron, transferrin, transferrin saturation, ferritin, caeruloplasmin, bleomycin detectable ('free') iron (BDI), and thiobarbituric acid reacting substances (TBARS) made over the first 28 days. Thirty infants remained oxygen dependent at 36 weeks postmenstrual age and were termed as having CLD. They were significantly lighter and less mature at birth and received more than twice as many transfusions during the 1st month. They had significantly lower transferrin levels initially but similar total iron and transferrin saturations as non-CLD infants. Ferritin and caeruloplasmin levels rose to significantly higher levels over the 1st month in CLD infants, and ferritin levels were significantly related to the number of transfusions given. Infants with higher ferritin levels were more likely to show BDI, although this was not associated with increased lipid peroxidation as evidenced by higher TBARS. CONCLUSION: It is unlikely that oxidative injury from ODFRs induced by blood transfusion contributes to the risk of developing CLD in preterm infants.

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