Pregnancy outcome in women with polycystic ovary syndrome in relation to second-trimester testosterone levels

Research questionDo women with polycystic ovary syndrome (PCOS) have higher testosterone levels during pregnancy and what role does high testosterone play in the development of obstetric complications?DesignRetrospective cohort study from Uppsala University Hospital, Sweden. The study population consisted of women with PCOS (n = 159) and a comparison group of women without PCOS matched for body mass index (n = 320). Plasma testosterone levels were measured in the early second trimester by liquid chromatography with tandem mass spectrometry, and women with PCOS were grouped into tertiles according to their testosterone levels. Possible associations with obstetric complications, maternal metabolic factors and offspring birth weight were explored by multivariable logistic and linear regression models.ResultsCompared with women who do not have PCOS, women with PCOS had higher total testosterone (median 1.94, interquartile range [IQR] 1.21–2.64 versus 1.41, IQR 0.89–1.97; P < 0.001), and free androgen index (median 0.25, IQR 0.15–0.36 versus 0.18, IQR 0.11–0.28; P < 0.001). Women with PCOS who had the highest levels of testosterone had increased risk for preeclampsia, even when adjusted for age, parity, country of birth and smoking (adjusted OR 6.16, 95% CI 1.82 to 20.91). No association was found between high testosterone in women with PCOS and other obstetric complications.ConclusionsWomen with PCOS have higher levels of total testosterone and free androgen index during pregnancy than women without PCOS matched for body mass index. Preliminary evidence shows that women with PCOS and the highest maternal testosterone levels in early second trimester had the highest risk of developing preeclampsia. This finding, however, is driven by a limited number of cases and should be interpreted with caution.     

MicroRNAs and their role for T stage determination and lymph node metastasis in early colon carcinoma

Worldwide, colon cancer is among the most common cancer entities. Understanding the molecular background is the key to enable accurate stage determination, which is crucial to assess optimal therapy options. The search for preoperative biomarkers is ongoing. In recent years, several studies have proposed a diagnostic and prognostic role for miRNAs in cancer. Aim of this study was to evaluate miRNA expression patterns correlating with tumor stage, especially lymph node metastasis, in primary colon carcinoma tissue. Screening was accomplished using GeneChip^® miRNA v3.0 arrays (Thermo Fisher Scientific, Waltham, MA, USA) and validated via TaqMan^® qPCR assays (Thermo Fisher Scientific, Waltham, MA, USA) to investigate miRNA expressions in 168 FFPE and 83 fresh frozen colon carcinoma samples. Regarding lymph node status, analyses displayed no significantly differential miRNA expression. Interestingly, divergent expression of miR-18a-5p, miR-20a-5p, miR-21-5p, miR-152-3p and miR-1973 was detected in stage pT1. Although miRNAs might not represent reliable biomarkers regarding lymph node metastasis status, they could support risk assessment in stage T1 tumors.     

Retinoblastoma and mental retardation microdeletion syndrome: clinical characterization and molecular dissection using array CGH

We describe three patients with retinoblastoma, dysmorphic features and developmental delay. Patients 1 and 2 have high and broad forehead, deeply grooved philtrum, thick anteverted lobes and thick helix. Patient 1 also has dolicocephaly, sacral pit/dimple and toe crowding; patient 2 shows intrauterine growth retardation and short fifth toe. Both patients have partial agenesis of corpus callosum. Patient 3 has growth retardation, microcephaly, thick lower lip and micrognathia. Using array-comparative genomic hybridization (CGH), we identified a 13q14 de novo deletion in patients 1 and 2, while patient 3 had a 7q11.21 maternally inherited deletion, probably not related to the disease. Our results confirm that a distinct facial phenotype is related to a 13q14 deletion. Patients with retinoblastoma and malformations without a peculiar facial phenotype may have a different deletion syndrome or a casual association of mental retardation and retinoblastoma. Using array-CGH, we defined a critical region for mental retardation and dysmorphic features. We compared this deletion with a smaller one in a patient with retinoblastoma (case 4) and identified two distinct critical regions, containing 30 genes. Four genes appear to be good functional candidates for the neurological phenotype: NUFIP1 (nuclear fragile X mental retardation protein 1), HTR2A (serotonin receptor 2A), PCDH8 (prothocaderin 8) and PCDH17 (prothocaderin 17).     

Unbound Corneocyte Lipid Envelopes in 12R-Lipoxygenase Deficiency Support a Specific Role in Lipid-Protein Cross-Linking

Loss-of-function mutations in arachidonate lipoxygenase 12B (ALOX12B) are an important cause of autosomal recessive congenital ichthyosis (ARCI). 12R-lipoxygenase (12R-LOX), the protein product of ALOX12B, has been proposed to covalently bind the corneocyte lipid envelope (CLE) to the proteinaceous corneocyte envelope, thereby providing a scaffold for the assembly of barrier-providing, mature lipid lamellae. To test this hypothesis, an in-depth ultrastructural examination of CLEs was performed in ALOX12B^−/− human and Alox12b^−/− mouse epidermis, extracting samples with pyridine to distinguish covalently attached CLEs from unbound (ie, noncovalently bound) CLEs. ALOX12B^−−/− stratum corneum contained abundant pyridine-extractable (ie, unbound) CLEs, compared with normal stratum corneum. These unbound CLEs were associated with defective post-secretory lipid processing, and were specific to 12R-LOX deficiency, because they were not observed with deficiency of the related ARCI-associated proteins, patatin-like phospholipase 1 (Pnpla1) or abhydrolase domain containing 5 (Abhd5). These results suggest that 12R-LOX contributes specifically to CLE–corneocyte envelope cross-linking, which appears to be a prerequisite for post-secretory lipid processing, and provide insights into the pathogenesis of 12R-LOX deficiency in this subtype of ARCI, as well as other conditions that display a defective CLE.

The corneocyte lipid envelope (CLE) (Figure 1D) is a lipid membrane composed of ω-hydroxy ceramides and ω-hydroxy fatty acids that are covalently bound to the extracellular surface of the proteinaceous corneocyte envelope (CE).^1,2 Investigation of autosomal recessive congenital ichthyosis (ARCI), caused by mutations in enzymes that contribute to CLE and CE synthesis, has revealed that the CLE-CE complex is required for skin barrier function.3, 4, 5, 6 Altered CLE-CE composition and structure are also associated with barrier dysfunction in many other skin conditions, including atopic dermatitis⁷ and psoriasis.⁸Open in a separate windowFigure 1Unbound corneocyte lipid envelopes (CLEs) in ALOX12B^−/− autosomal recessive congenital ichthyosis (ARCI). A: The current model of CLE biogenesis involves esterified ω-hydroxy-acyl sphingosine (EOS) synthesis catalyzed by patatin-like phospholipase 1 (PNPLA1) and abhydrolase domain containing 5 (ABHD5), transport to the plasma membrane (not shown), and EOS oxidation catalyzed sequentially by 12R-lipoxygenase, epidermal lipoxygenase 3, and short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7). A subsequent reaction between EOS–epoxy enone (EpO) and peptides of the corneocyte envelope (CE) is thought to occur nonenzymatically to generate protein-bound ω-hydroxy-acyl sphingosine (OS), which may subsequently be hydrolyzed by ceramidases (C''ase) to form bound ω-hydroxy fatty acids (OHFA). B: A 53-year–old male ARCI patient with homozygous deletion/in-frame mutations (c.786_788delCTT leading to amino acid loss p. Phe263del) in ALOX12B. Note the accentuated skin lines (black arrow) and erythroderma that are typical findings in retinoid-treated ARCI. C–F: Skin samples from an ARCI-unaffected human subject (C and D) or from the ALOX12B^−/− patient (E and F) were divided and processed for electron microscopy with or without pyridine (Pyr) extraction, as indicated before fixation to extract unbound lipids (osmium post-fix). D–F: Covalently bound CLEs were visualized in the Pyr-extracted, ARCI-unaffected sample (D), whereas CLEs in the ALOX12B^−/− sample seemed to be unbound because they were removed by Pyr extraction (E and F). G: Quantification of CLE abundance. Black arrowheads indicate CEs; white arrowheads, CLEs; white arrows, corneodesmosomes (shown to demonstrate that images are in focus). Data show the means ± SEM (G). n = 10 randomly acquired high-powered fields used for quantification in each determination (G). ∗P < 0.05, ∗∗∗P < 0.001. Scale bars = 100 nm (C–F). C, corneocyte; C18:2-TAG, linoleoyl triacylglycerol; EpOH, epoxy alcohol; OOH, hydroperoxide.The major unbound lipid precursor of the CLE is most likely esterified ω-hydroxy-acyl sphingosine (EOS; an abundant ω-O-acyl ceramide in mammalian epidermis). In the current model (Figure 1A), the final step of EOS synthesis is transfer of linoleate (C18:2) from triglyceride to ω-hydroxy-acyl sphingosine (the ceramide ω-hydroxy-acyl sphingosine), catalyzed by patatin-like phospholipase 1 (PNPLA1) and its coactivator abhydrolase domain containing 5 (ABHD5)/comparative gene identification-58 (CGI-58).^4,9, 10, 11 EOS and glucosylated EOS are then stored and transported in lamellar bodies,¹² whose limiting membranes fuse with the plasma membrane during lipid secretion in the outer stratum granulosum at the site of the developing CLE.⁵ Subsequently, oxidation reactions catalyzed sequentially by 12R-lipoxygenase (12R-LOX; encoded by the gene ALOX12B), epidermal lipoxygenase 3 (encoded by ALOXE3), and short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) are proposed to transform the pentadiene group in the ω-linoleate of EOS into an epoxy enone that can react nonenzymatically with peptides on the external face of the CE (especially involucrin, envoplakin, and periplakin¹³) to form protein-bound ω-hydroxy-acyl sphingosine.^14,15 The bound ω-hydroxy fatty acids could be formed either by a similar sequence starting with ω-O-acyl fatty acids instead of EOS¹⁶ or via hydrolysis of the amide bond of bound ω-hydroxy-acyl sphingosine by ceramidases.¹⁷This model explains why both CLEs and their ω-hydroxy ceramide and ω-hydroxy fatty acid constituents are diminished in ARCI with defects in EOS synthesis^4,5 or EOS oxidation. The phenotype associated with these two subgroups of ARCI is indistinguishable,¹⁸ consistent with the possibility that the disease features are a consequence of the shared CLE defect. However, one major difference is that unbound EOS is reduced in EOS synthesis defects, but normal or increased in EOS oxidation defects (the metabolic changes associated with these differences are illustrated) (Supplemental Figure S1).^19,20 Some of this excess EOS could be contained within abnormal CLEs that fail to be cross-linked to protein (referred to as unbound CLEs throughout this article) due to the defect in EOS oxidation, although these have not yet been reported. The identification of such unbound CLEs would support a specific role for 12R-LOX in CLE-CE cross-linking and could provide insight into CLE biogenesis and the potential treatment of CLE defects in ichthyosis and other conditions. Therefore, in the current study, a detailed ultrastructural examination was performed on epidermis from an ALOX12B^−/− ARCI patient and from Alox12b^−/− mice, using the amphipathic solvent pyridine to distinguish unbound from bound CLEs.     

Low frequency of H-ras mutations in hepatocellular adenomas and carcinomas and in hepatoblastomas from B6C3F1 mice exposed to oxazepam in the diet

Oxazepam has been the subject of recent toxicological and carcinogenesisstudies because it is a commonly prescribed tranquilizer andhas been shown to cause tumors in rodents. In this study, maleand female B6C3F1 mice receIved 0,125, 2500 or 5000 p.p.m. oxazepamin the diet for up to 2 years. Hepatocellular adenomas and carcinomas,as well as hepatoblastomas, which developed in these mice, wereexamined for the presence of activated ms proto-oncogenes. DNAwas Isolated from 20 or more tumors from each exposure groupand analyzed by oligonudeotide hybridiza tion, single-strandedconformation polymorphism analysis and direct sequencing ofPCR-ampllfied H-ms gene fragments for codon 61 mutations. Thirteenof 37 (35%) hepatocellular adenomas and carcinomas from the125 p.p.m. exposure group had mutations in codon 61, while mutationswere detected in only 2 of 25 or 8% of the liver tumors fromthe 2500 p.p.m. exposure group and none of the 22 tumors fromthe 5000 p.p.m. group. This compares to 63% of 126 historIcalcontrol liver tumors and 55% of 20 liver tumors from unexposedB6C3F1 mice in this study. In addition, 12 hepatoblastomas fromthe two high dose groups were examined for H-ras mutations atcodon 61, but none were detected. No tumor DNAs from any ofthe exposure groups tested had mutations In codons 12, 13 or117 of the H-ras gene or codons 12 or 13 of the K-ras gene,the other known hotspots for ras activation in mouse liver tumors.These results, together with those from the National ToxicologyProgram study showing no evidence of cytotoxicity or genotoxicityby oxazepam, suggest that oxazepam preferentially promotes cellsthat have activating lesions other than ras.     

Hepatocarcinogenicity of chlordane in B6C3F1 and B6D2F1 male mice: evidence for regression in B6C3F1 mice and carcinogenesis independent of ras proto-oncogene activation

Logistic regression analysis of age-specific prevalences forneoplastic and non-neoplastic liver lesions was used to examinetreatment responses for B6C3F1 and B6D2F1 male mice continuouslyexposed to chlordane (55 p.p.m.) and to determine whether neoplasmswere dependent on continuous exposure in the B6C3F1 mice. Inorder to determine if ras oncogene activation plays a role inthe carcinogenicity of chlordane and whether the activationis dependent on genetic background, liver tumors from chlordane-treatedB6C3F1 and B6D2F1 mice were analyzed for the presence of activatingmutations in the ras oncogene. The overall liver tumor prevalenceat terminal killing was nearly 100% for both strains; however,the age-specific prevalence increased more rapidly in B6C3F1mice than in B6D2F1 mice. Tumor-bearing B6C3F1 mice had an averageof two more tumors per liver than B6D2F1 mice at their respectiveterminal killings (5.4 versus 3.3). When chlordane exposurewas discontinued for a group of B6C3F1 mice (‘stop’group) at 491 days of age, overall tumor multiplicity significantlydecreased by 30% from an average of 4.4 per tumor-bearing-animalat 525 days to 3.1 at terminal killing (568 days). Over thesame time period the prevalence of hepatocellular carcinomassignificantly decreased from 80 to 54% and adenomas from 100to 93% by terminal killing in B6C3F1 ‘stop-group’mice. Chlordane induced diffuse hepatocellular centrilobularhypertrophy, frequent multinucleate hepatocytes, toxic changeand hepatoproliferative lesions composed predominantly of acidophilichepatocytes in nearly 100% of both the B6C3F1 and B6D2F1 mice.The development of histological evidence of toxicity closelyparalleled the temporal development of hepatocellular neoplasiaand decreased in severity when the tumor burden was maximal.No H- or K-ras mutations were detected in the chlordane-inducedhepatocellular tumors in B6C3F1 mice (15 adenomas and 15 carcinomas)or B6D2F1 mice (10 adenomas and 10 carcinomas). In conclusion,chlordane induced liver tumors in both B6C3F1 and B6D2F1 malemice by mechanisms independent of ras oncogene activation and30% of both benign and malignant liver tumors in the B6C3F1mice regressed after exposure was discontinued.     

High frequency of K-ras mutations in spontaneous and vinyl carbamate-induced lung tumors of relatively resistant B6CF1 (C57BL/6JxBALB/cJ) mice

The murine K-ras proto-oncogene is hypothesized to be a pulmonaryadenoma susceptibility gene. This postulate is supported bythe previous demonstration of a preference for mutation of theK-ras allele from the susceptible parent in lung tumors of A/JxC3HF1 mice. We have examined K-ras activation in control and vinylcarbamate (VC) (single dose 0.03 µmol/g i.p.) treatedB6CF1 mice, the progeny of resistant C57BL/6J and intermediatelysensitive BALB/cJ parents. Thirty-four of 37 tumors from VC-treatedmice and 17 of 23 from controls contained activating K-ras weresimilar for the two groups, except that 7 tumors from VC-treatedmice had A     

In vivo evidence for increased oxidation of circulating LDL in impaired glucose tolerance

Oxidized LDL (oxLDL) is a key mediator in atherogenesis and a marker of coronary artery disease (CAD). Type 2 diabetes is associated with excessive cardiovascular morbidity and mortality. Because atherogenesis starts before diabetes is diagnosed, we investigated whether circulating oxLDL levels are increased in impaired glucose tolerance (IGT). OxLDL levels were measured in 376 subjects with normal glucose tolerance (NGT), 113 patients with IGT, and 54 patients with newly diagnosed type 2 diabetes. After correction for age and BMI, serum levels of oxLDL were significantly increased in IGT versus NGT subjects (P = 0.002). OxLDL levels were not associated with the following parameters of the oxidative/antioxidative balance in the blood: total antioxidant capacity, urate-to-allantoin ratio, and circulating phagocyte oxygenation activity. In stepwise multivariate analysis, LDL cholesterol (P < 0.0005) and triglycerides (P < 0.0005) were the strongest predictors of circulating oxLDL levels, followed by HDL cholesterol (P = 0.003), 2-h postchallenge C-peptide (P = 0.011), fasting free fatty acids (P = 0.013), and serum paraoxonase activity (P = 0.035). The strong correlation of oxLDL with LDL cholesterol and triglycerides indicates that LDL oxidation in IGT is preferentially associated with dyslipidemia. OxLDL increase may explain the high atherogenic potency of dyslipidemia in the prediabetic state.