Functional and clinical impact of novel TMPRSS6 variants in iron-refractory iron-deficiency anemia patients and genotype-phenotype studies.

Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation analysis demonstrates that patients carrying two nonsense mutations present a more severe anemia and microcytosis and higher hepcidin levels than the other patients. We confirm that TMPRSS6 mutations are spread along the gene and that mechanistically they fully or partially abrogate hepcidin inhibition. Genotyping IRIDA patients help in predicting IRIDA severity and may be useful for predicting response to iron treatment.

Endoscopic full-thickness biopsy of the gastric wall with defect closure by using an endoscopic suturing device: survival porcine study.

BACKGROUND: The pathogenesis of several common gastric motility diseases and functional GI disorders remains essentially unexplained. Gastric wall biopsies that include the muscularis propria to evaluate the enteric nervous system, interstitial cells of Cajal, and immune cells can provide important insights for our understanding of the etiology of these disorders. OBJECTIVES: To determine the technical feasibility, reproducibility, and safety of performing a full-thickness gastric biopsy (FTGB) by using a submucosal endoscopy with mucosal flap (SEMF) technique; the technical feasibility, reproducibility, and safety of tissue closure by using an endoscopic suturing device; the ability to identify myenteric ganglia in resected specimens; and the long-term safety. DESIGN: Single center, preclinical survival study. SETTING: Animal research laboratory, developmental endoscopy unit. SUBJECTS: Twelve domestic pigs. INTERVENTIONS: Animals underwent an SEMF procedure with gastric muscularis propria resection. The resultant offset mucosal entry site was closed by using an endoscopic suturing device. Animals were kept alive for 2 weeks. MAIN OUTCOME MEASUREMENTS: The technical feasibility, reproducibility, and safety of the procedure; the clinical course of the animals; the histological and immunochemical evaluation of the resected specimen to determine whether myenteric ganglia were present in the sample. RESULTS: FTGB was performed by using the SEMF technique in all 12 animals. The offset mucosal entry site was successfully closed by using the suturing device in all animals. The mean resected tissue specimen size was 11 mm. Mean total procedure time was 61 minutes with 2 to 4 interrupted sutures placed per animal. Histology showed muscularis propria and serosa, confirming full-thickness resections in all animals. Myenteric ganglia were visualized in 11 of 12 animals. The clinical course was uneventful. Repeat endoscopy and necropsy at 2 weeks showed absence of ulceration at both the mucosal entry sites and overlying the more distal muscularis propria resection sites. There was complete healing of the serosa in all animals with minimal single-band adhesions in 5 of 12 animals. Retained sutures were present in 10 of 12 animals. LIMITATIONS: Animal experiment. CONCLUSIONS: FTGB by using the SEMF technique and an endoscopic suturing device is technically feasible, reproducible, and safe. Larger tissue specimens will allow improved analysis of multiple cell types.

Natural orifice transluminal endoscopic surgery for patients with perforated peptic ulcer.

BACKGROUND: Perforation accounts for 70% of deaths attributed to peptic ulcers. Laparoscopic repair is effective but infrequently used. Our aim was to assess how many patients with perforated peptic ulcer could be candidates for a transluminal endoscopic omental patch closure. METHODS: This retrospective study reviewed patients with perforated peptic ulcer from 2005 to 2010. Demographics, ulcer characteristics, operative procedure, and outcomes were recorded. Candidates for endoscopic transluminal repair were defined as those having undergone omental patch closure of an ulcer of appropriate size and no contraindications to laparoscopy or endoscopy. RESULTS: In the retrospective review, a total of 104 patients were identified; 62% female, mean age = 68 years, mean ASA of 3, and 63% medication-related ulcers. Fifty-nine (63%) had an omental patch (80% open), and 35 (37%) had other procedures. Ten patients had nonoperative management. Thirty-day mortality was 14% and 1 year mortality was 35%. Forty-nine patients (52%) were considered potential candidates for transluminal repair. CONCLUSION: Sixty-three percent of our patients sustained a medication-related perforation with 1 year mortality of 35%. The majority of patients were treated using open omental patch repair. Transluminal endoscopic repair may provide an additional situation for a minimally invasive approach for a number of these patients.

A comparative study of endoscopic full-thickness and partial-thickness myotomy using submucosal endoscopy with mucosal safety flap (SEMF) technique.

BACKGROUND: Esophageal myotomy using submucosal endoscopy with mucosal safety flap (SEMF) has been proposed as a new treatment of achalasia. In this technique, a partial-thickness myotomy (PTM) preserving the longitudinal outer esophageal muscular layer is advocated, which is different from the usual full-thickness myotomy (FTM) performed surgically. The aim of this study was to compare endoscopic FTM and PTM and analyze the outcomes of each method after a 4 week survival period. METHODS: Twenty-four pigs were randomly assigned into group A (FTM, 12 animals) and group B (PTM) to undergo endoscopic myotomy. Lower esophageal sphincter (LES) pressure was assessed using pull-through manometry. For statistical analysis we compared the average esophageal sphincter pressure values at baseline, after 2 weeks, and after 4 weeks between groups A and B. The P value was set as <0.05 for significance. RESULTS: Eighteen animals were included for statistical analysis. Mean (SD) LES pressures were similar between groups A and B (nine animals each) at baseline [group A = 23 (10.4) mmHg; group B = 20.7 (8.7) mmHg; P = 0.79], after 2 weeks [group A = 19 (7.7) mmHg; group B = 21.8 (8.4) mmHg; P = 0.79], and after 4 weeks [group A = 22.6 (10.2) mmHg; group B = 20.7 (9) mmHg; P = 0.82]. LES pressures were significantly reduced in three animals after 4 weeks: one animal (1%) in group A and two animals (2.5%) in group B. An extended myotomy (3 cm below the cardia) was achieved in three animals and was responsible for the significant drop in LES pressure seen in the two animals from group B. CONCLUSION: Esophageal myotomy using SEMF is a feasible yet challenging procedure in pigs. Full-thickness myotomy does not seem to be superior to partial-thickness myotomy as demonstrated by pull-through manometry. Endoscopic esophageal myotomy results are greatly influenced by obtaining adequate myotomy extension into the gastric cardia.

Missense SLC25A38 variations play an important role in autosomal recessive inherited sideroblastic anemia.

BACKGROUND: Congenital sideroblastic anemias are rare disorders with several genetic causes; they are characterized by erythroblast mitochondrial iron overload, differ greatly in severity and some occur within a syndrome. The most common cause of non-syndromic, microcytic sideroblastic anemia is a defect in the X-linked 5-aminolevulinate synthase 2 gene but this is not always present. Recently, variations in the gene for the mitochondrial carrier SLC25A38 were reported to cause a non-syndromic, severe type of autosomal-recessive sideroblastic anemia. Further evaluation of the importance of this gene was required to estimate the proportion of patients affected and to gain further insight into the range and types of variations involved. DESIGN AND METHODS: In three European diagnostic laboratories sequence analysis of SLC25A38 was performed on DNA from patients affected by congenital sideroblastic anemia of a non-syndromic nature not caused by variations in the 5-aminolevulinate synthase 2 gene. RESULTS: Eleven patients whose ancestral origins spread across several continents were homozygous or compound heterozygous for ten different SLC25A38 variations causing premature termination of translation (p.Arg117X, p.Tyr109LeufsX43), predicted splicing alteration (c.625G>C; p.Asp209His) or missense substitution (p.Gln56Lys, p.Arg134Cys, p.Ile147Asn, p.Arg187Gln, p.Pro190Arg, p.Gly228Val, p.Arg278Gly). Only three of these variations have been described previously (p.Arg117X, p.Tyr109LeufsX43 and p.Asp209His). All new variants reported here are missense and affect conserved amino acids. Structure modeling suggests that these variants may influence different aspects of transport as described for mutations in other mitochondrial carrier disorders. CONCLUSIONS: Mutations in the SLC25A38 gene cause severe, non-syndromic, microcytic/hypochromic sideroblastic anemia in many populations. Missense mutations are shown to be of importance as are mutations that affect protein production. Further investigation of these mutations should shed light on structure-function relationships in this protein.

Pulmonary and peritoneal inflammatory findings in transgastric NOTES compared with laparoscopy: pooled analysis from randomized porcine survival studies.

BACKGROUND: Laparoscopy, which is a minimally invasive surgery, is associated with decreased peritoneal adhesions and inflammatory response compared with laparotomy. OBJECTIVE: To evaluate whether natural orifice transluminal endoscopic surgery (NOTES) leads to an attenuated peritoneal response compared with laparoscopy. DESIGN: Pooled histologic analysis from 2 randomized porcine trials. SETTING: Laboratory. INTERVENTION: Histologic analysis of swine undergoing diagnostic laparoscopy, diagnostic NOTES peritoneoscopy, NOTES with transgastric mesh placement, or diagnostic endoscopy (no gastrotomy) followed by laparoscopic mesh placement. MAIN OUTCOME MEASUREMENTS: The presence and grade of inflammation in necropsy specimens of lung, liver, and spleen as reviewed by a blinded veterinary pathologist. RESULTS: Four NOTES mesh animals exhibited mesh infections at necropsy. Tissue from 48 swine were available for analysis. Pulmonary inflammation, liver fibrosis, and spleen capsulitis were the primary findings. No difference was seen in the incidence of each finding among groups. The severity of the pulmonary inflammation in the laparoscopy group was significantly higher than in the NOTES groups. The NOTES mesh group exhibited significantly more severe liver fibrosis and spleen capsulitis. There was no difference between clinical behavior, serum white blood cell count, or peritoneal white blood cell count among groups in either study. Intra-abdominal pressures during NOTES were lower than during laparoscopy. LIMITATIONS: Pooled analysis of 2 separate studies. CONCLUSION: More severe pulmonary inflammation was found in animals undergoing longer laparoscopic procedures with higher intra-abdominal pressures. Intraperitoneal inflammation was most significant with transgastric mesh placement, likely caused by infections.

Randomized study of natural orifice transluminal endoscopic surgery and endoscopy shows similar hemodynamic impact in a porcine model.

BACKGROUND: A previous porcine study showed a significant difference in heart rate and diastolic blood pressure (DBP) between natural orifice transluminal endoscopic surgery (NOTES) and laparoscopy. This study evaluated the hemodynamics during endoscopy, laparoscopy, and transluminal access. METHODS: For this study, 37 female swine were randomized and invasively monitored in terms of blood and abdominal pressure, heart rate, and arterial blood gas (ABG) during 90-min procedures. Group 1 (n = 11) underwent NOTES peritoneoscopy; group 2 (n = 14) underwent 45-min diagnostic endoscopy, a 10-min washout period, and 35-min laparoscopy with mesh placement; and group 3 (n = 12) NOTES had transgastric mesh placement. The groups were compared using a mixed model and a Spearman trend test. This study was approved by Institutional Animal Care and Use Committee (IACUC). RESULTS: No difference in the systolic blood pressure (SBP) was noted. During the initial 30 min, DBP increased significantly from baseline in groups 1 (p < 0.001) and 2 (p = 0.01), but not in group 3 (p = 0.08). The mean DBP did not differ between the groups. During laparoscopy, the average end-tidal carbon dioxide (CO(2)) level was 6.6 mmHg higher in group 2 than in group 1 (p = 0.01). The heart rate and ABG values did not differ between the groups (p ≥ 0.10). CONCLUSION: Heart rate and DBP were similar for NOTES and endoscopy. The differences seen in a previous trial comparing NOTES and laparoscopy were not duplicated. The initial DBP increased for the endoscopy and diagnostic NOTES animals. Differences in end-tidal CO(2) were encountered again during the shortened laparoscopy segment.

Patients' experience of oxygen therapy and dyspnea: a qualitative study in home palliative care.

BACKGROUND: Dyspnea is a common and distressing symptom in advanced cancer patients. Our preliminary work shows that in the home palliative care population sampled for this study, the prevalence of dyspnea is 29.5% and of those, 26.2% used oxygen therapy. Previous studies suggested that oxygen therapy can be a burden to patients. PURPOSE: This study seeks to report the prevalence and describe the experience of dyspnea, pattern of oxygen use, and patients' perceived benefits and/or burdens of oxygen therapy in home palliative care patients receiving oxygen therapy. METHODS: Qualitative in-depth interviews, using an interview guide, were conducted with eight participants in their homes. Thematic analysis was performed using a framework approach. RESULTS: All patients in this project used oxygen most of the time. The descriptions of shortness of breath varied and were nonspecific. The patients identified more advantages than disadvantages. The advantages of oxygen use included increased functional capacity, patients' perceiving oxygen as a life-saving intervention, as well as a symptom-management tool. The identified disadvantages were decreased mobility, discomfort related to the nasal prongs, barriers to accessing oxygen therapy and noise related to the equipment. CONCLUSION: The advantages of oxygen usage outweighed the disadvantages for this sample of patients in the home palliative setting.

Preliminary performance of a flexible cap and catheter-based endoscopic suturing system.

BACKGROUND: Translation of natural orifice transluminal endoscopic surgery (NOTES) into clinical applications requires efficient and reliable enterotomy closure. OBJECTIVE: To evaluate a prototype endoscopic suturing system for enterotomy closure. DESIGN: This study took place in an ex vivo animal laboratory. Isolated porcine stomachs were contained within a plastic molded abdominal torso. The device specifications included a curved needle, end-cap assembly with a side-mounting wire-actuation channel, a needle-exchange assembly that operates within an endoscopic working channel, and a detachable needle tip attached to suture material. INTERVENTIONS: Mucosal templates (3-cm circular markings) for targeted suture placement were created along the anterior wall of the stomach (cardia, antrum, and body). Device performance and functionality were studied in 3 ways: suture placement, purse-string closure, and edge-to-edge gastrotomy closure. Interrupted and running stitches were placed with the endoscope straight and retroflexed. Simple leak testing was conducted. RESULTS: Sutures could accurately be placed at preset templated markings. Creation of a purse-string gastrotomy closure confirmed the capability to place a set of circumferential full-thickness running sutures during a single endoscopic intubation that resulted in a leak-proof closure. Edge-to-edge full-thickness tissue apposition was accomplished, which provided a water-tight closure of an 18-mm gastrotomy. The device worked consistently, without any problems. CONCLUSIONS: This endoscopic suturing device provided accurate placement of full-thickness sutures during a single intubation and permitted satisfactory tissue apposition. Standardized leak testing is needed for further development and evaluation of new devices. The catheter-driven needle actuator and the transfer-component system were intuitive and universally adaptable to any endoscope. This closure device may advance transluminal therapies by offering a secure, efficient method of hollow viscus closure.

The tailless nuclear receptor acts as a dedicated repressor in the early Drosophila embryo.

Tailless is an orphan nuclear receptor that controls terminal body patterning in Drosophila. Genetic analyses have revealed both positive and negative regulatory interactions of Tailless with various target genes, leading to the idea that, like many other nuclear receptors, Tailless mediates both activation and repression of transcription. In this paper, we have examined the consequences of converting Tailless into an obligate repressor and compared the activities of the resulting protein with those of wild-type Tailless. We find that this repressor form of Tailless behaves like the intact protein in gain- and loss-of-function experiments, being sufficient to support normal embryonic development and establish accurate patterns of gene expression even for positive Tailless targets such as hunchback and brachyenteron. This suggests that Tailless functions exclusively as a transcriptional repressor in the embryo and that the observed positive interactions of Tailless with specific targets are secondary effects involving repression of repressors. We provide evidence that knirps is one such repressor gene acting between Tailless and its indirect positive targets. Finally, our results indicate that Tailless exerts an active mechanism of repression via its ligand-binding domain and that this activity is largely independent of the activation function 2 (AF2) motif characteristic of most nuclear receptors.

Anatomical considerations for natural orifice translumenal endoscopic surgery.

Success in surgical procedures relies on the surgeon's understanding of anatomy and the ways in which the internal organs relate to one another. Recently, a new surgical technique has been introduced. Natural orifice translumenal endoscopic surgery (NOTES) uses the body's natural orifices (mouth, anus, urethra, or vagina) as entrance points to the peritoneal cavities (through the stomach, rectum, bladder, or posterior vaginal fornix). NOTES techniques have proven feasible in both animal and early human trials. While it remains to be seen what advantages NOTES possesses over traditional surgical approaches, a clear understanding of human anatomy will be critical for successful, safe NOTES procedures. This article summarizes the development and the basic techniques of NOTES and reviews those anatomical considerations specific to NOTES.

Surgeons without scalpels. A review of natural orifice translumenal endoscopic surgery.

Natural orifice translumenal endoscopic surgery (NOTES) is a new technique that allows access to the peritoneal cavity without passing through the anterior abdominal wall. This article reviews the evolution of NOTES and describes how it is currently being evaluated in animal models. It also discusses the potential benefits of the technique as well as the challenges it faces.

Functional consequences of transferrin receptor-2 mutations causing hereditary hemochromatosis type 3.

Hereditary hemochromatosis (HH) type 3 is an autosomal recessive disorder of iron metabolism characterized by excessive iron deposition in the liver and caused by mutations in the transferrin receptor 2 (TFR2) gene. Here, we describe three new HH type 3 Spanish families with four TFR2 mutations (p.Gly792Arg, c.1606-8A>G, Gln306*, and Gln672*). The missense variation p.Gly792Arg was found in homozygosity in two adult patients of the same family, and in compound heterozygosity in an adult proband that also carries a novel intronic change (c.1606-8A>G). Two new nonsense TFR2 mutations (Gln306* and Gln672*) were detected in a pediatric case. We examine the functional consequences of two TFR2 variants (p.Gly792Arg and c.1606-8A>G) using molecular and computational methods. Cellular protein localization studies using immunofluorescence demonstrated that the plasma membrane localization of p.Gly792Arg TFR2 is impaired. Splicing studies in vitro and in vivo reveal that the c.1606-8A>G mutation leads to the creation of a new acceptor splice site and an aberrant TFR2 mRNA. The reported mutations caused HH type 3 by protein truncation, altering TFR2 membrane localization or by mRNA splicing defect, producing a nonfunctional TFR2 protein and a defective signaling transduction for hepcidin regulation. TFR2 genotyping should be considered in adult but also in pediatric cases with early-onset of iron overload.

Novel mutations in the ferritin-L iron-responsive element that only mildly impair IRP binding cause hereditary hyperferritinaemia cataract syndrome.

BACKGROUND: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant disease characterized by increased serum ferritin levels and early onset of bilateral cataract. The disease is caused by mutations in the Iron-Responsive Element (IRE) located in the 5' untranslated region of L-Ferritin (FTL) mRNA, which post-transcriptionally regulates ferritin expression. METHODS: We describe two families presenting high serum ferritin levels and juvenile cataract with novel mutations in the L-ferritin IRE. The mutations were further characterized by in vitro functional studies. RESULTS: We have identified two novel mutations in the IRE of L-Ferritin causing HHCS: the Badalona +36C > U and the Heidelberg +52 G > C mutation. Both mutations conferred reduced binding affinity on recombinant Iron Regulatory Proteins (IPRs) in EMSA experiments. Interestingly, the Badalona +36C > U mutation was found not only in heterozygosity, as expected for an autosomal dominant disease, but also in the homozygous state in some affected subjects. Additionally we report an update of all mutations identified so far to cause HHCS. CONCLUSIONS: The Badalona +36C > U and Heidelberg +52 G > C mutations within the L-ferritin IRE only mildly alter the binding capacity of the Iron Regulatory Proteins but are still causative for the disease.

A single nucleotide polymorphism in STK11 influences insulin sensitivity and metformin efficacy in hyperinsulinemic girls with androgen excess.

OBJECTIVE: Serine-threonine kinase STK11 catalyzes the AMP-activated protein kinase complex. We tested the hypothesis that a gene variant in STK11 contributes to variation in insulin sensitivity and metformin efficacy. RESEARCH DESIGN AND METHODS: We studied the effects of a single nucleotide polymorphism (SNP) (rs8111699) in STK11 on endocrine-metabolic and body composition indexes before and after 1 year of metformin in 85 hyperinsulinemic girls with androgen excess, representing a continuum from prepuberal girls with a combined history of low birth weight and precocious pubarche over to postmenarchial girls with hyperinsulinemic ovarian hyperandrogenism. Metformin was dosed at 425 mg/day in younger girls and 850 mg/day in older girls. STK11 rs8111699 was genotyped. Endocrine-metabolic features were assessed in the fasting state; body composition was estimated by absorptiometry. RESULTS: Genotype effects were similar in younger and older girls. At baseline, the mutated G allele in STK11 rs8111699 was associated with higher insulin and IGF-I levels (both P < 0.005). The response to metformin differed by STK11 genotype: GG homozygotes (n = 24) had robust metabolic improvements, GC heterozygotes (n = 38) had intermediate responses, and CC homozygotes (n = 23) had almost no response. Such differences were found for 1-year changes in body composition, circulating insulin, IGF-I, free androgen index, and lipids (all P < 0.005). CONCLUSIONS: In hyperinsulinemic girls with androgen excess, the STK11 rs8111699 SNP influences insulin sensitivity and metformin efficacy, so that the girls with the least favorable endocrine-metabolic profile improve most with metformin therapy.

Natural orifice translumenal endoscopic surgery used for perforated viscus repair is feasible using lower peritoneal pressures than laparoscopy in a porcine model.

BACKGROUND: Procedure-related complications contribute to 1-year mortality in patients with perforated ulcers. Natural orifice translumenal endoscopic surgery (NOTES) might offer a new repair approach. STUDY DESIGN: Swine were randomized to laparoscopic or NOTES repair. Laparoscopic gastrotomy creation (1 cm) was followed by 4 hours soilage time. After peritoneal cavity irrigation (per group assignment), repair proceeded with a laparoscopic or NOTES approach. For NOTES repair, omentum was endoscopically grasped, pulled into the gastric lumen, and fixed with metallic clips. Feasibility; time to complete procedures; pneumoperitoneal pressures; and clinical parameters, including necropsy and peritoneal culture at 2 weeks, were recorded. RESULTS: NOTES repair failed in 1 animal (technical); repair was completed laparoscopically, and data were analyzed as intention to treat. Specific NOTES repair time (minutes) was comparable with laparoscopy (36 versus 46; p = 0.2). Mean abdominal pressure (mmHg) required to complete NOTES repair was lower than in laparoscopy (4 versus 12; p < 0.001). Nineteen of 23 animals thrived until necropsy at 2 weeks. Three animals succumbed to airway compromise in recovery; 1 NOTES animal failed to thrive on postoperative day 7. No intra-abdominal cause for these deaths was found. At necropsy all repairs were intact, and peritoneal cultures revealed a small and equivalent amount of colony-forming units in each group. CONCLUSIONS: Endoscopic ulcer repair appears technically feasible with similar clinical and infectious outcomes to laparoscopy. The lower required pneumoperitoneal pressures used in these NOTES techniques are recognizable different outcomes from laparoscopy and can be advantageous in critically ill patients.

Retinoid-dependent mRNA expression and poly-(A) contents in bovine oocytes meiotically arrested and/or matured in vitro.

The presence of retinoic acid (RA) during in vitro maturation (IVM) improves bovine oocyte quality and developmental potential. In this work, we investigated the underlying molecular mechanisms. Cumulus-oocyte complexes were meiotically arrested by roscovitine and/or matured in defined medium containing RA, 1% ethanol (vehicle), or no additives. Cumulus-free oocytes were analyzed for poly-(A) mRNA contents and relative mRNA expression of genes involved in cell cycle regulation (cyclin B1 and H1) and antioxidative defence (Mn-superoxide dismutase and glucose-6-phosphate dehydrogenase). Poly-(A) mRNA increased after meiotic inhibition and decreased with IVM completion, both in meiotically arrested and permissively matured oocytes, i.e., matured without previous meiotic arrest. RA dramatically increased poly-(A) mRNA in meiotically arrested oocytes, but more than half of the poly-(A) mRNA disappeared during maturation. Irrespective of oocyte origin, transcripts were detected for all the genes analyzed. IVM, with or without previous meiotic inhibition, increased expression of cyclin B1 and glucose-6-phosphate dehydrogenase, and decreased cyclin H1 and Mn-superoxide dismutase. Except for a decreasing of Mn-superoxide dismutase in meiotically arrested and matured oocytes, RA did not affect mRNA expression. Ethanol led to an abnormal poly-(A) mRNA profile and expression of all the genes analyzed. RA does not modify expression of cyclin B1 and HI genes in the bovine oocyte, and probably does not generate oxidative stress. In addition, RA enhanced mRNA amount as measured by poly-(A) mRNA contents.