The change in endometrial thickness following progesterone exposure correlates with in vitro fertilization outcome after transfer of vitrified-warmed blastocysts.

PURPOSE: To determine if the change in endometrial thickness following exogenous progesterone (P) initiation correlates with outcome following autologous transfer of a single thawed blastocyst. METHODS: The study is a retrospective observational cohort study conducted at a private fertility center. Patients scheduled for thawed blastocyst transfer received artificial endometrial preparation (artificial cycle FET) and underwent serial ultrasonography. The main outcomes were the rate of ongoing pregnancy (fetal heart motion at 12 weeks of gestation) and early pregnancy loss. Logistic regression was used to test for correlations between these outcomes and the change in endometrial thickness while adjusting for potential confounders (patient age, embryo quality, and the use of genetic testing). RESULTS: There were 232 qualifying autologous single-blastocyst transfers in the 20-month study period ending 31 December 2019. Mean endometrial thicknesses were 3.8 mm, 10.0 mm, and 11.2 mm at baseline, P initiation, and at transfer, respectively. The change in endometrial thickness after exogenous P exposure ranged from - 5 to + 9 mm and negatively correlated with ongoing pregnancy in logistic regression analyses. Specifically, ongoing pregnancy rates per transfer were 63.2% in 19 cases where endometria compacted by 10% or more, 64.2% in 95 cases where there was unchanged endometrial thickness, and 52.5% in 118 cases where endometria expanded. CONCLUSIONS: The change in endometrial thickness after P initiation was associated with the probability of ongoing pregnancy but not with early pregnancy loss. Ongoing pregnancy rates were greater in endometria with negative growth (compaction) when compared to endometria that grew (expanded) after P exposure.

The risk of embryo-endometrium asynchrony increases with maternal age after ovarian stimulation and IVF.

This retrospective cohort analysis examined the effects of maternal age on the incidence of factors associated with embryo-endometrium asynchrony in fresh autologous blastocyst transfer. The study included 1169 routine fresh autologous blastocyst transfers. The main outcome measure was asynchronous transfer defined by delayed (day 6) blastocyst transfer or elevated pre-ovulatory serum progesterone level. Compared with patients younger than 35 years, patients 35 years or older had increased risk of having at least one risk factor for asynchronous transfer, including premature progesterone elevation or delayed blastocyst transfer (RR 1.36; 95% CI 1.24 to 1.50). The older group had increased risk of simultaneously having both risk factors (RR 1.61, 95% CI 1.17 to 2.21) compared with the younger group. In patients younger than 35 years, live birth rate per transfer was 62.9% with day 5 transfer and low progesterone, declining to 27.9% for day 6 transfer combined with elevated progesterone. In patients 35 years or older, live birth rate per transfer was 38.0% with day 5 transfer and low progesterone, declining to 18.1% for day 6 transfer combined with elevated progesterone. Indicators of embryo-endometrium asynchrony increase in prevalence as women age and asynchrony disproportionately decreases birth rates in older patients.

Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfers in high responders.

Clinical pregnancy rates of 80% and 65% were observed in cycles using thawed and fresh embryos, respectively, although embryo quality indicators revealed morphologically and numerically inferior embryo cohorts after cryopreservation. Subsequent logistic regression analysis controlled for differences in embryo quality and revealed significantly greater probability of clinical pregnancy with thawed embryos when compared with fresh, suggesting a negative effect of ovarian stimulation on endometrial receptivity.

Evidence of impaired endometrial receptivity after ovarian stimulation for in vitro fertilization: a prospective randomized trial comparing fresh and frozen-thawed embryo transfer in normal responders.

OBJECTIVE: To compare success rates between fresh ETs after ovarian stimulation and frozen-thawed ETs (FET) after artificial endometrial preparation, to compare endometrial receptivity. DESIGN: Randomized, controlled trial. SETTING: Private fertility center. PATIENT(S): There were 53 patients completing fresh blastocyst transfer (fresh group) and 50 patients completing FET (cryopreservation group). All were first-time IVF patients aged <41 years, with cycle day 3 FSH <10 mIU/mL and 8-15 antral follicles. INTERVENTION(S): Randomized to fresh or thawed ET. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate per transfer. RESULT(S): The clinical pregnancy rate per transfer was 84.0% in the cryopreservation group and 54.7% in the fresh group. The implantation rates were 70.8% and 38.9%, respectively. The ongoing pregnancy rates per transfer (at 10 weeks' gestation) were 78.0% and 50.9%, respectively. The attributable risk percentage of implantation failure due to reduced endometrial receptivity in the fresh group was 64.7%. CONCLUSION(S): The clinical pregnancy rate per transfer was significantly greater in the cryopreservation group than in the fresh group. These results strongly suggest impaired endometrial receptivity in fresh ET cycles after ovarian stimulation, when compared with FET cycles with artificial endometrial preparation. Impaired endometrial receptivity apparently accounted for most implantation failures in the fresh group. ClinicalTrials.gov Identifier: NCT00963625.

Comparison of "triggers" using leuprolide acetate alone or in combination with low-dose human chorionic gonadotropin.

This retrospective study of fresh autologous blastocyst transfers in high responders compared ongoing pregnancy rates in cycles that followed trigger with GnRH agonist (GnRHa) alone with standard luteal support, GnRHa alone with enhanced luteal support, or GnRHa with concomitant low-dose hCG (dual trigger). Ongoing pregnancy rates were significantly increased with the dual trigger or with enhanced luteal support, whereas the incidence of clinically significant ovarian hyperstimulation syndrome was 0.0% in the groups receiving only GnRHa and 0.5% (1 of 182) in patients receiving GnRHa with concomitant low-dose hCG.

Efficacy of induced luteinizing hormone surge after "trigger" with gonadotropin-releasing hormone agonist.

The magnitude of the LH surge after GnRH agonist "trigger" was correlated with oocyte yield and maturity and was suboptimal in approximately half of the cycles. A modest reduction in oocyte yield and maturity was observed when the serum level of LH 12 hours after GnRH agonist trigger was less than the median value (52 IU/L), and a dramatic reduction in yield and maturity was observed when that level was less than 12 IU/L.

Embryo cryopreservation rescues cycles with premature luteinization.

OBJECTIVE: To determine whether embryo cryopreservation in cycles with elevated preovulatory P followed by thaw, extended culture, and transfer results in greater ongoing pregnancy rates than fresh blastocyst transfer. DESIGN: Retrospective matched cohort study. SETTING: Private fertility center. PATIENT(S): The study group consisted of 118 consecutive thaws of bipronucleate (2PN) oocytes derived from autologous cycles with elevated preovulatory P, resulting in 95 blastocyst transfers. The control group was selected by matching on the number of 2PN oocytes and patient age and consisted of 118 fresh cycles with elevated preovulatory P, including 108 fresh autologous blastocyst transfers. All patients were <41 years old at the time of stimulation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation and ongoing pregnancy rates. RESULT(S): The study group had significantly fewer blastocysts per 2PN oocyte than the control group (19.7% vs. 36.8%, respectively) and a significantly greater cancellation rate (19.5% vs. 8.5%, respectively). However, the ongoing pregnancy rate per cycle was significantly higher in the study group than in the control group (56.8% vs. 32.2%, respectively), resulting from greater rates of implantation (56.8% vs. 26.9%, respectively) and of ongoing pregnancy per transfer (70.5% vs. 35.2%, respectively). CONCLUSION(S): In cycles with elevated preovulatory P, the probabilities of implantation and ongoing pregnancy are increased if all 2PN oocytes are cryopreserved and subsequently thawed and cultured to the blastocyst stage before transfer.

Contrasting patterns in in vitro fertilization pregnancy rates among fresh autologous, fresh oocyte donor, and cryopreserved cycles with the use of day 5 or day 6 blastocysts may reflect differences in embryo-endometrium synchrony.

OBJECTIVE: To compare the effect of day 5 and day 6 blastocyst transfers on patterns of implantation rates and pregnancy rates (PRs) among fresh autologous, oocyte donor, and frozen ET (FET) cycles. DESIGN: Retrospective study. SETTING: Private fertility center. PATIENT(S): The study included 377 fresh autologous cycles, 106 autologous FET cycles, and 56 fresh oocyte donor cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Implantation rates and clinical PRs. RESULT(S): The clinical PR for day 5 blastocyst transfers was higher than for day 6 blastocyst transfers in fresh autologous cycles (PRs, 51.0% and 33.3%, respectively). However, there was no significant difference between transfers of blastocysts cryopreserved on day 5 and day 6 in FET cycles (PRs, 63.6% and 58.9%, respectively). Furthermore, day 6 blastocyst transfers significantly outperformed day 5 transfers in donor cycles (PRs, 63.0% and 86.2%, respectively), a reversal of the pattern seen in the fresh autologous cycles. Day 6 blastocysts were associated with a significantly greater PR in FET cycles than in fresh autologous cycles (58.9% and 33.3%, respectively). CONCLUSION(S): The superior PRs with day 5 blastocyst transfers in fresh autologous cycles and with day 6 blastocysts in donor cycles may have resulted from better synchrony with endometrial development. This was further supported by the superior performance of day 6 blastocysts in FET cycles relative to their fresh counterparts. Similar PRs with cryopreserved day 5 and day 6 blastocysts in FET cycles may reflect that, in these cycles, day 5 and day 6 blastocysts had equivalent quality and similar synchrony with the endometrium.

Large blastocyst diameter, early blastulation, and low preovulatory serum progesterone are dominant predictors of clinical pregnancy in fresh autologous cycles.

OBJECTIVE: To identify dominant predictors of clinical pregnancy in IVF cycles. DESIGN: Retrospective study. SETTING: Private fertility center. PATIENT(S): The study included 580 fresh autologous IVF cycles with blastocyst transfer. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate. RESULT(S): A set of 25 suspected predictors was used to develop predictive models of clinical pregnancy in a set of 361 blastocyst transfer cycles. Initial bivariate analysis identified 14 of these variables that were significant enough to be candidate variables for multiple logistic regression. Similar sets of significant variables were identified by using alternative approaches for model construction. The final model included blastocyst diameter, day of blastulation, and preovulatory serum P level as significant predictors of clinical pregnancy. Specifically, clinical pregnancy was predicted by preovulatory serum P of <1.0 ng/mL, blastulation on day 5, and large blastocyst diameter. Of these variables, blastocyst diameter was the most significant predictor of clinical pregnancy in the multivariate models. The final model was validated against a separate set of 219 subsequent blastocyst transfer cycles. CONCLUSION(S): Pre-ovulatory serum P level, blastulation day, and embryo diameter are simultaneously predictive of clinical pregnancy, and their relationships with clinical pregnancy are consistent with an effect of embryo-endometrium synchrony.

Gonadotropin-releasing hormone agonist combined with a reduced dose of human chorionic gonadotropin for final oocyte maturation in fresh autologous cycles of in vitro fertilization.

Acceptable rates of fertilization, implantation, clinical pregnancy, ongoing pregnancy, and early pregnancy loss were achieved in high responders after triggering final oocyte maturation with a combination of leuprolide acetate and hCG (1,000 to 2,500 IU). These findings, along with the absence of ovarian hyperstimulation syndrome, suggest that this dual trigger is safe and effective for oocyte maturation in patients with significant risk factors for ovarian hyperstimulation syndrome.

Comparison of human chorionic gonadotropin and gonadotropin-releasing hormone agonist for final oocyte maturation in oocyte donor cycles.

In this retrospective study of 74 oocyte-donor IVF cycles, the rates of fertilization, implantation, clinical pregnancy, ongoing pregnancy, and early pregnancy loss were similar after an agonist or hCG trigger. These findings suggest that the agonist trigger is a viable alternative for oocyte donors with significant risk factors for ovarian hyperstimulation syndrome.

Acute and selective regulation of glyceroneogenesis and cytosolic phosphoenolpyruvate carboxykinase in adipose tissue by thiazolidinediones in type 2 diabetes.

AIMS/HYPOTHESIS: Regulation of glyceroneogenesis and its key enzyme cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) plays a major role in the control of fatty acid release from adipose tissue. Here we investigate the effect of rosiglitazone on the expression of genes involved in fatty acid metabolism and the resulting metabolic consequences. MATERIALS AND METHODS: Rosiglitazone was administered to Zucker fa/fa rats for 4 days and to 24 diabetic patients for 12 weeks, then mRNA expression for the genes encoding PEPCK-C, mitochondrial PEPCK, adipocyte lipid-binding protein, glycerol kinase, lipoprotein lipase and glycerol-3-phosphate dehydrogenase was examined in s.c. adipose tissue by real-time RT-PCR. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. Cultured adipose tissue explants from overweight women undergoing plastic surgery were incubated with rosiglitazone for various times before mRNA determination and analysis of PEPCK-C protein, activity and glyceroneogenesis. RESULTS: Rosiglitazone administration to rats induced the expression of the gene encoding PEPCK-C mRNA (PCK1) and PEPCK-C activity in adipose tissue with a resulting 2.5-fold increase in glyceroneogenesis. This was accompanied by an improvement in dyslipidaemia as demonstrated by the decrease in plasma NEFAs and triacylglycerol. In rosiglitazone-treated diabetic patients, PCK1 mRNA was raised 2.5-fold in s.c. adipose tissue. Rosiglitazone treatment of adipose tissue explants from overweight women caused a selective augmentation in PCK1 mRNA which reached a maximum of 9-fold at 14 h, while mRNA for other genes remained unaffected. Experiments with inhibitors showed a direct and transcription-only effect, which was followed by an increase in PEPCK-C protein, enzyme activity and glyceroneogenesis. CONCLUSIONS/INTERPRETATION: These results favour adipocyte glyceroneogenesis as the initial thiazolidinedione-responsive pathway leading to improvement in dyslipidaemia.

Removing tetrahedra from manifold tetrahedralisation: application to real-time surgical simulation.

This paper proposes an efficient method for removing tetrahedra from a tetrahedral mesh while keeping its manifold property. We first define precisely the notion of manifold tetrahedral mesh and stress its relevance in the context of real-time surgery simulation. We then provide a method for removing a tetrahedron that complies with the manifold definition. This removal may require in some cases the removal of neighboring tetrahedra. After providing an exhaustive description of the tetrahedron removal algorithm, its efficiency is evaluated for different mesh configurations. This algorithm is currently used in the context of real-time surgery simulation where the action of an ultrasonic lancet can be simulated by the removal of small set of tetrahedra from a tetrahedralisation.

A single element in the phosphoenolpyruvate carboxykinase gene mediates thiazolidinedione action specifically in adipocytes.

Phosphoenolpyruvate carboxykinase (PEPCK) is the key enzyme in glyceroneogenesis, an important metabolic pathway that functions to restrain the release of non-esterified fatty acids (NEFAs) from adipocytes. The antidiabetic drugs known as thiazolidinediones (TZDs) are thought to achieve some of their benefits by lowering elevated plasma NEFAs. Moreover, peroxisome proliferator activated receptor gamma (PPARgamma) mediates the antidiabetic effects of TZDs, though many TZD responses appear to occur via PPARgamma-independent pathways. PPARgamma is required for adipocyte PEPCK expression, hence PEPCK could be a major target gene for the antidiabetic actions of TZDs. Here we used tissue culture and transfection assays to confirm that the TZD, rosiglitazone, stimulates PEPCK gene transcription specifically in adipocytes. We made the novel observation that this effect was by far the most rapid and robust among several other genes expressed in adipocytes. Adipocytes were transfected with a PEPCK/chloramphenicol acetyltransferase chimeric gene, in which either of the two previously discovered PPARgamma/retinoid X receptor alpha response elements, PCK2 and gAF1/PCK1, had been inactivated by mutagenesis. We demonstrate that PCK2 alone is a bona fide thiazolidinedione response element. We show also that the regulation of PEPCK by PPARs is cell-specific and isotype-specific since rosiglitazone induces PEPCK gene expression selectively in adipocytes, and PPARalpha- and PPARbeta-specific activators are inefficient. Hence, TZDs could lower plasma NEFAs via PPARgamma and PEPCK by enhancing adipocyte glyceroneogenesis.

Formation of glycine receptor clusters and their accumulation at synapses.

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor alpha subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an alpha mutant subunit bearing the gephyrin-binding motif of the beta subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR beta subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.

Peroxisome proliferator activated receptor-gamma, leptin and tumor necrosis factor-alpha mRNA expression during very low calorie diet in subcutaneous adipose tissue in obese women.

BACKGROUND: PPAR gamma, leptin and TNF alpha are three major factors that play a key role in influencing adipocyte differentiation and both adipose tissue function and metabolism. However, the regulation of these three genes during a dynamic period of weight loss is unknown. We therefore investigated the concomitant regulation of the mRNA expression of PPAR gamma, leptin and TNF alpha in adipose tissue during a 21-day very low calorie diet (VLCD) in 12 non-diabetic obese women. METHODS: The mRNA levels of PPAR gamma, leptin and TNF alpha were quantified by quantitative RT-competitive PCR in abdominal subcutaneous adipose tissue before and during VLCD (940 kcal/day). RESULTS: VLCD induced weight loss (approximately 6 kg) and improved insulin sensitivity. Simultaneously, VLCD induced the reduction in the adipose tissue mRNA abundances of PPAR gamma (-13%, p < 0.05) and of leptin (-58%, p < 0.005), whereas TNF alpha mRNA levels increased (+78%, p < 0.005). PPAR gamma and leptin mRNA levels were correlated before (r = 0.778, p < 0.01) and after VLCD (r = 0.797, p < 0.01). Serum HDL-cholesterol concentrations were positively associated with PPAR gamma (r = 0.696, p < 0.03) and leptin (r = 0.806, p < 0.01) mRNA levels. CONCLUSIONS: The increase in TNF alpha mRNA levels suggested that a local increased expression of this cytokine in adipose tissue might play a role in the control of the fat mass during weight loss. PPAR gamma and leptin mRNA levels were positively associated both before and after VLCD, suggesting that common regulatory mechanism(s) might control their expression. More strikingly, we found strong positive correlations between circulating HDL-cholesterol and both PPAR gamma and leptin mRNA levels, suggesting the existence of physiological links between circulating lipoprotein metabolism and adipose tissue function.

Identification of an adipocyte-specific negative glucose response region in the cytosolic aspartate aminotransferase gene.

Cytosolic aspartate aminotransferase (cAspAT) participates in gluconeogenesis in the liver and is expected to exert a glyceroneogenic function in the adipose tissue when the supply of glucose is limited. Here we demonstrate that adipose cAspAT messenger RNA (mRNA) is increased when rats are fed a low carbohydrate diet. In the 3T3-F442A, BFC-1 adipocyte cell lines and differentiated adipocytes in primary culture, a 24 h glucose deprivation induces approximately a 4-fold increase in cytosolic AspAT (cAspAT) mRNA, whereas mitochondrial AspAT mRNA remains unchanged. cAspAT activity is also increased in a weaker but reproducible manner. Addition of glucose within a physiological range of concentrations reverses the increase of cAspAT mRNA in 8 h (EC50 = 1.25 g/liter). Such a regulation requires protein synthesis and is specific for adipocytes differentiated in culture. It does not occur in Fao or H4IIE hepatoma cells, in C2 muscle cells, or in 293 kidney cells. 2-deoxyglucose mimicks glucose, while 3-orthomethyl-glucose has no effect, suggesting that glucose-6-phosphate is the effector. cAspAT mRNA stability is not affected by glucose deprivation. To ascertain the transcriptional nature of the glucose effect, we have stably transfected 3T3-F442A adipoblasts with constructs containing the chloramphenicol acetyltransferase reporter gene under the control of either 5'-deletions of the cAspAT gene promoter or internal fragments in an heterologous context. We demonstrate that a glucose response element(s) is present in the region between -1838 and -1702 bp relative to the translation start site. In this region, three DNA sequences bind nuclear proteins from adipocytes as shown by footprinting experiments. Our results indicate that cAspAT gene transcription is repressed by glucose selectively in adipocytes.

Mechanism of adenovirus improvement of cationic liposome-mediated gene transfer.

Substantial effort has been focused on the development of highly efficient gene transfer strategies. Although viral and non-viral methods have been elaborated, mechanisms of gene delivery are still poorly understood. We exploited our recent observation that replication-deficient type 5 adenovirus dramatically enhances lipofectAMINE-mediated gene transfer (lipoadenofection) in differentiated cells to elucidate the mechanism of adenovirus action in this process. Heat-induced denaturation of viral capsid abolishes adenovirus action whereas inactivation of viral genome by short treatment with UV has no effect. Electron microscopic observations reveal the formation of a complex containing adenovirus and lipofectAMINE which probably carries DNA into cells via endocytosis. Anti-adenovirus antiserum or monoclonal anti-alpha(v)beta3 integrin antibody inhibits lipoadenofection, at least partially. Neutralization of endosomal compartments with chloroquine, ammonium chloride or monensin does not prevent adenovirus improvement of gene transfer. Hence, adenovirus-lipofectAMINE-DNA complexes in which viral particles are each encompassed by three lipid layers, penetrate cells via an endocytic pathway involving probably the adenovirus receptor and alpha(v)beta3 integrin. The resulting efficient transfer and expression of plasmid DNA proceeds from a mechanism in which adenoviral endosomolytic activity appears to be required while viral genome is not essential.

Adenovirus enhancement of polyethylenimine-mediated transfer of regulated genes in differentiated cells.

Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of adenovirus in a PEI-DNA-adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI-adenofection.

Glucocorticoids use a positive liver element to repress fibrate-induced adipose transcription of the phosphoenolpyruvate carboxykinase gene.

Glucocorticoids inhibit basal and hormone-induced phosphoenolpyruvate carboxykinase (PEPCK) gene transcription in adipocytes whereas beta-adrenergic agonists and fibrates are stimulatory. Here we show that dexamethasone inhibits the induction of PEPCK mRNA by isoprenaline or clofibrate in 3T3-F442A adipocytes. RU 38486 antagonizes dexamethasone effect, suggesting the involvement of the glucocorticoid receptor. In H4IIE hepatoma cells, glucocorticoids enhance PEPCK gene transcription through a complex region which encompasses an element, AF1, with a direct repeat 1-type sequence. Mutations in the AF1 sequence abolish binding of nuclear factors from liver and from 3T3-F442A adipocytes. We transiently transfected 3T3-F442A cells with a wild type or an AF1-mutated PEPCK-CAT construct comprising -2100 to +69 base pairs of the promoter fused to the chloramphenicol acetyltransferase (CAT) gene. With both constructs, CAT activity is decreased by dexamethasone and is increased by isoprenaline or by clofibrate. However, dexamethasone is unable to inhibit clofibrate induction of CAT activity in cells transfected with the AF1-mutated construct whereas it prevents isoprenaline action on both constructs. Hence, although a single hormone can repress stimulations originating from different intracellular routes, sites in the promoter which mediate inhibition of a specific stimulation are distinct.