Delphine Haouzi

Hepatotoxicity due to mitochondrial dysfunction.

Mitochondria are involved in fatty acid β-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation, which provide most of the cell energy. Mitochondria are also the main source of reactive oxygen species in the cell and are involved in cell demise through opening of the mitochondrial permeability transition pore. It was therefore to be expected that mitochondrial dysfunction could be a major mechanism of drug-induced liver disease. Microvesicular steatosis (which may cause liver failure, coma, and death) is the consequence of severe impairment of mitochondrial β-oxidation. Endogenous compounds (such as cytokines or female sex hormones) or xenobiotics (including toxins such as ethanol and drugs such as aspirin, valproic acid, ibuprofen, or zidovudine) can inhibit β-oxidation directly or through a primary effect on the mitochondrial genome or the respiratory chain itself. In some patients, infections and cytokines, or inborn errors of β-oxidation enzymes or the mitochondrial genome, may favor the appearance of drug-induced microvesicular steatosis. Nonalcoholic steatohepatitis may develop under conditions causing prolonged, microvesicular, and/or macrovacuolar steatosis. In this condition, chronic impairment of mitochondrial β-oxidation (causing steatosis) and the respiratory chain (increasing the production of ROS) lead to lipid peroxidation, which, in turn, may cause the diverse lesions of steatohepatitis, namely, necrosis, inflammation, Mallorys bodies, and fibrosis. Finally, mitochondria are involved in several forms of drug-induced cytolytic hepatitis, through inhibition or uncoupling of respiration or through a drug-induced or reactive metabolite-induced mitochondrial permeability transition. The latter effect commits hepatocytes to either apoptosis or necrosis, depending on the number of organelles that have undergone the permeability transition.

A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

An alcoholic binge causes massive degradation of hepatic mitochondrial DNA in mice

BACKGROUND & AIMS Ethanol causes oxidative stress in the hepatic mitochondria of experimental animals and mitochondrial DNA deletions in alcoholics. We postulated that ethanol intoxication may cause mitochondrial DNA strand breaks. METHODS Effects of an intragastric dose of ethanol (5 g/kg) on hepatic mitochondrial DNA levels, structure, and synthesis were determined by slot blot hybridization, Southern blot hybridization, and in vivo [3H]thymidine incorporation, respectively. RESULTS Two hours after ethanol administration, ethane exhalation (an index of lipid peroxidation) increased by 133%, although hepatic lipids were unchanged. Mitochondrial DNA was depleted by 51%. Its supercoiled form disappeared, whereas linearized forms increased. Long polymerase chain reaction evidenced lesions blocking polymerase progress on the mitochondrial genome. Mitochondrial transcripts decreased. Subsequently, [3H]thymidine incorporation into mitochondrial DNA increased, and mitochondrial DNA levels were restored. In contrast, nuclear DNA was not fragmented and its [3H]thymidine incorporation was unchanged. Liver ultrastructure only showed inconstant mitochondrial lesions. Ethanol-induced mitochondrial DNA depletion was prevented by 4-methylpyrazole, an inhibitor of ethanol metabolism, and attenuated by melatonin, an antioxidant. CONCLUSIONS After an alcoholic binge, ethanol metabolism causes oxidative stress and hepatic mitochondrial DNA degradation in mice. DNA strand breaks may be involved in the development of mitochondrial DNA deletions in alcoholics.

Identification of new biomarkers of human endometrial receptivity in the natural cycle

BACKGROUND Identification of new markers assessing endometrial receptivity may help in improving the clinical outcome of IVF. This study aimed at identifying genes expressed in human endometrium during the implantation window that could be used as such markers. METHODS A series of normoresponder patients (n = 31) underwent endometrial biopsies (n = 62, 2 per patient) during the early secretory phase, 2 days after the LH surge (LH + 2) and the mid-secretory phase (LH + 7) of the same natural cycle that preceded a new ICSI attempt for male infertility factor. Samples were analyzed using DNA microarrays and gene expression profiles at the time of the implantation window were computed. Systems biology analysis allowed the identification of biological pathways that were over-represented in this signature. A new approach for class prediction applied to microarray experiments was then used to identify biomarkers putatively involved in endometrial receptiveness. RESULTS Five genes expressed during the implantation window were all up-regulated in the LH + 7 samples compared with LH + 2 [laminin beta3 (P = 0.002), microfibril-associated protein 5 (P = 0.009), angiopoietin-like 1 (P = 0.005), endocrine gland-derived vascular endothelial growth factor (P = 0.049) and nuclear localized factor 2 (P = 0.007)]. Increased expression was validated by quantitative RT-PCR. CONCLUSIONS Five genes have been identified for the first time as being up-regulated during the implantation window and are proposed as new biomarkers for exploration of endometrial receptiveness. As the endometrial biopsy procedure can be performed during a natural cycle, it would be worth testing this approach as a novel strategy in patients with poor implantation after IVF or ICSI.

Expression of the liver Na+-independent organic anion transporting polypeptide (oatp-1) in rats with bile duct ligation

BACKGROUND/AIMS In rats with cholestasis due to bile duct ligation, the expression of the Na+-dependent taurocholate co-transporting polypeptide, the major uptake system for conjugated bile acids in hepatocytes, is down-regulated. Our purpose was to examine the expression of the organic anion transporting polypeptide, a Na+-independent uptake system for bile acids and organic anions, in rats with bile duct ligation, and to compare the expression of organic anion transporting polypeptide to that of Na+-dependent taurocholate co-transporting polypeptide. METHODS Rats with bile duct ligation were studied after 1, 3 or 7 days. The expression of organic anion transporting polypeptide and Na+-dependent taurocholate co-transporting polypeptide proteins was examined by Western blot analysis and steady-state mRNA levels were determined by Northern blot analysis using cDNAs encoding organic anion transporting polypeptide and Na+-dependent taurocholate co-transporting polypeptide. Sham-operated animals were used as controls. RESULTS The expression of organic anion transporting polypeptide protein was slightly, but not significantly, decreased 1 day after ligation (10.3%); it was markedly decreased after 3 days (56.9%; p<0.03) and 7 days (46.8%; p<0.05) compared to sham-operated animals. Steady-state mRNA levels of organic anion transporting polypeptide were decreased by 79.7% (p<0.04), 48.8% (p<0.02) and 57.4% (p<0.02) after 1, 3 and 7 days respectively. For comparison, Na+-dependent taurocholate co-transporting polypeptide protein and mRNA levels were decreased by 73.8% (p<0.03) and 70.0% (p<0.05) at 1 day and remained low after 3 and 7 days. CONCLUSIONS In rats with bile duct ligation, the expression of organic anion transporting polypeptide protein and mRNA is down-regulated. Down-regulation of organic anion transporting polypeptide seems less pronounced than that of Na+-dependent taurocholate co-transporting polypeptide. Nevertheless, it could contribute to a decreased uptake of potentially toxic bile acids or organic anions in this situation.

Mechanisms for experimental buprenorphine hepatotoxicity: major role of mitochondrial dysfunction versus metabolic activation

BACKGROUND/AIMS Although sublingual buprenorphine is safely used as a substitution drug in heroin addicts, large overdoses or intravenous misuse may cause hepatitis. Buprenorphine is N-dealkylated to norbuprenorphine by CYP3A. METHODS We investigated the mitochondrial effects and metabolic activation of buprenorphine in isolated rat liver mitochondria and microsomes, and its toxicity in isolated rat hepatocytes and treated mice. RESULTS Whereas norbuprenorphine had few mitochondrial effects, buprenorphine (25-200 microM) concentrated in mitochondria, collapsed the membrane potential, inhibited beta-oxidation, and both uncoupled and inhibited respiration in rat liver mitochondria. Both buprenorphine and norbuprenorphine (200 microM) underwent CYP3A-mediated covalent binding to rat liver microsomal proteins and both caused moderate glutathione depletion and increased cell calcium in isolated rat hepatocytes, but only buprenorphine also depleted cell adenosine triphosphate (ATP) and caused necrotic cell death. Four hours after buprenorphine administration to mice (100 nmol/g body weight), hepatic glutathione was unchanged, while ATP was decreased and serum transaminase increased. This transaminase increase was attenuated by a CYP3A inducer and aggravated by a CYP3A inhibitor. CONCLUSIONS Both buprenorphine and norbuprenorphine undergo metabolic activation, but only buprenorphine impairs mitochondrial respiration and ATP formation. The hepatotoxicity of high concentrations or doses of buprenorphine is mainly related to its mitochondrial effects.

Dynamic changes in gene expression during human early embryo development: from fundamental aspects to clinical applications

BACKGROUND The first week of human embryonic development comprises a series of events that change highly specialized germ cells into undifferentiated human embryonic stem cells (hESCs) that display an extraordinarily broad developmental potential. The understanding of these events is crucial to the improvement of the success rate of in vitro fertilization. With the emergence of new technologies such as Omics, the gene expression profiling of human oocytes, embryos and hESCs has been performed and generated a flood of data related to the molecular signature of early embryo development. METHODS In order to understand the complex genetic network that controls the first week of embryo development, we performed a systematic review and study of this issue. We performed a literature search using PubMed and EMBASE to identify all relevant studies published as original articles in English up to March 2010 (n = 165). We also analyzed the transcriptome of human oocytes, embryos and hESCs. RESULTS Distinct sets of genes were revealed by comparing the expression profiles of oocytes, embryos on Day 3 and hESCs, which are associated with totipotency, pluripotency and reprogramming properties, respectively. Known components of two signaling pathways (WNT and transforming growth factor-β) were linked to oocyte maturation and early embryonic development. CONCLUSIONS Omics analysis provides tools for understanding the molecular mechanisms and signaling pathways controlling early embryonic development. Furthermore, we discuss the clinical relevance of using a non-invasive molecular approach to embryo selection for the single-embryo transfer program.

Controlled Ovarian Hyperstimulation for In Vitro Fertilization Alters Endometrial Receptivity in Humans: Protocol Effects

The impact of gonadotropin-releasing hormone (GnRH) agonist long compared with GnRH antagonist protocols, under in vitro fertilization conditions on endometrial receptivity, is still debated. Therefore, we compared the effect of both GnRH antagonist and agonist long protocols on the endometrial receptivity by analyzing, to our knowledge for the first time, the global gene expression profile shift during the prereceptive and receptive stages of stimulated cycles under the two GnRH analogue protocols compared with natural cycles in the same patients. For the same normal-responder patients, endometrial biopsies were collected on the day of oocyte retrieval and on the day of embryo transfer after human chorionic gonadotropin administration of a stimulated cycle with either GnRH agonist long or GnRH antagonist protocols and compared with the prereceptive and receptive stages of a natural cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved during the prereceptive stage to the receptive endometrial transition of stimulated and natural cycles were analyzed and compared for each patient. Both protocols affect endometrial receptivity in comparison with their natural cycle in the same patients. Major differences in endometrial chemokines and growth factors under stimulated cycles in comparison with natural cycles were observed. Such an effect has been associated with gene expression alterations of endometrial receptivity. However, the endometrial receptivity under the GnRH antagonist protocol was more similar to the natural cycle receptivity than that under the GnRH agonist protocol.

MicroRNAs: new candidates for the regulation of the human cumulus–oocyte complex

STUDY QUESTION What is the expression pattern of microRNAs (miRNAs) in human cumulus-oocyte complexes (COCs)? SUMMARY ANSWER Several miRNAs are enriched in cumulus cells (CCs) or oocytes, and are predicted to target genes involved in biological functions of the COC. WHAT IS KNOWN ALREADY The transcriptional profiles of human MII oocytes and the surrounding CCs are known. However, very limited data are available about post-transcriptional regulators, such as miRNAs. This is the first study focussing on the identification and quantification of small RNAs, including miRNAs, in human oocytes and CCs using a deep-sequencing approach. STUDY DESIGN, SIZE, DURATION MII oocytes and CCs were collected from women who underwent IVF. PARTICIPANTS/MATERIALS, SETTING, METHODS Using the Illumina/deep-sequencing technology, we analyzed the small RNAome of pooled MII oocytes (n = 24) and CC samples (n = 20). The mRNA targets of CC and MII oocyte miRNAs were identified using in silico prediction algorithms. Using oligonucleotide microarrays, genome-wide gene expression was studied in oocytes (10 pools of 19 ± 3 oocytes/each) and 10 individual CC samples. TaqMan miRNA assays were used to confirm the sequencing results in independent pools of MII oocytes (3 pools of 8 ± 3 oocytes/each) and CC samples (3 pools of 7 ± 3 CCs/each). The functional role of one miRNA, MIR23a, was assessed in primary cultures of human CCs. MAIN RESULTS AND THE ROLE OF CHANCE Deep sequencing of small RNAs yielded more than 1 million raw reads. By mapping reads with a single location to the human genome, known miRNAs that were abundant in MII oocytes (MIR184, MIR100 and MIR10A) or CCs (MIR29a, MIR30d, MIR21, MIR93, MIR320a, MIR125a and the LET7 family) were identified. Predicted target genes of the oocyte miRNAs were associated with the regulation of transcription and cell cycle, whereas genes targeted by CC miRNAs were involved in extracellular matrix and apoptosis. Comparison of the predicted miRNA target genes and mRNA microarray data resulted in a list of 224 target genes that were differentially expressed in MII oocytes and CCs, including PTGS2, CTGF and BMPR1B that are important for cumulus-oocyte communication. Functional analysis using primary CC cultures revealed that BCL2 and CYP19A1 mRNA levels were decreased upon MIR23a overexpression. LIMITATIONS, REASONS FOR CAUTION Only known miRNAs were investigated in the present study on COCs. Moreover, the source of the material is MII oocytes that failed to fertilize. WIDER IMPLICATIONS OF THE FINDINGS The present findings suggest that miRNA could play a role in the regulation of the oocyte and CC crosstalk. STUDY FUNDING/COMPETING INTEREST(S) This work was partially supported by a grant from Ferring Pharmaceuticals. The authors of the study have no conflict of interest to report. TRIAL REGISTRATION NUMBER Not applicable.

Human Cumulus Cells Molecular Signature in Relation to Oocyte Nuclear Maturity Stage

The bi-directional communication between the oocyte and the surrounding cumulus cells (CCs) is crucial for the acquisition of oocyte competence. We investigated the transcriptomic profile of human CCs isolated from mature and immature oocytes under stimulated cycle. We used human Genome U133 Plus 2.0 microarrays to perform an extensive analysis of the genes expressed in human CCs obtained from patients undergoing intra-cytoplasmic sperm injection. CC samples were isolated from oocyte at germinal vesicle, stage metaphase I and stage metaphase II. For microarray analysis, we used eight chips for each CC category. Significance analysis of microarray multiclass was used to analyze the microarray data. Validation was performed by RT-qPCR using an independent cohort of CC samples. We identified differentially over-expressed genes between the three CC categories. This study revealed a specific signature of gene expression in CCs issued from MII oocyte compared with germinal vesicle and metaphase I. The CC gene expression profile, which is specific of MII mature oocyte, can be useful as predictors of oocyte quality.