Human sperm decondensation in vitro is related to cleavage rate and embryo quality in IVF.

PURPOSE: To investigate whether the ability of human spermatozoa to decondense in vitro in the presence of heparin (Hep) and glutathione (GSH) is related to assisted reproduction (ART) success. METHODS: Cross-sectional pilot study involving male partners of 129 infertile couples undergoing ICSI with (45) or without (84) donor oocytes at two infertility clinics in CABA, Argentina, between October 2012 and December 2013. In vitro decondensation kinetics with Hep and GSH and DNA fragmentation (TUNEL) were determined on the same sample used for ICSI. The possible relationship of decondensation parameters (maximum decondensation and decondensation velocity) and TUNEL values with ART success was evaluated. RESULTS: Embryo quality correlated positively with decondensation velocity (D60/D30) (Spearman's correlation, p < 0.05). According to D60/D30 values, patients were classified as slow decondensers (SlowD) (n = 68) or fast decondensers (FastD) (n = 61). Embryo quality was better in FastD (unpaired t test, p < 0.05). FastD and SlowD were subdivided according to use of donor oocytes. Among SlowD, biochemical and clinical pregnancy rates per transfer were significantly higher in donor (n = 19) vs. in non-donor (n = 31) cycles (Fisher's exact test, p < 0.05). TUNEL values were not related to embryo quality, but no clinical pregnancies or live births were achieved in TUNEL+ SlowD (n = 7). CONCLUSION: Decondensation kinetics of human spermatozoa in vitro with Hep and GSH could be related to embryo quality and ART success.

Root caries analysis in working population of 35-44 years of age (Spain).

BACKGROUND: The aim of this study was to analyse the influence of socio-demographic variables, toothbrushing frequency, frequency of snacking between meals, and tobacco and alcohol consumption, in root caries in the Spanish working population of Valencia and Murcia regions. MATERIAL AND METHODS: Cross sectional study of 458 workers 35-44 years of age, who underwent a routine work-related check-up, from June 2009 to April 2010, and were also examined, following the WHO methodology, by a calibrated dentist. Stratified random sampling. Participants fulfilled a questionnaire comprising demographic data, toothbrushing frequency, snacking frequency and tobacco and alcohol consumption. RESULTS: The DFS index (root caries) in the employed population of 35-44 years was 0.45 ± 1.3, with a root caries prevalence of 18.6% and an active root caries prevalence of 13.5%. Higher root caries prevalence and active root caries prevalence were associated with male gender, manual occupations, foreign country of origin, lower levels of education and income, lower brushing frequency and higher frequency of snacking between meals. The DFS index was associated with all studied socio-demographic variables, but gender, and it was also associated with brushing frequency. The mean number of root decayed teeth was associated with all socio-demographic variables, but country of origin, and it was also associated with brushing frequency. CONCLUSIONS: Adult workers 35-44 years of age showed worse root condition in regard to caries than general population of this age cohort. In this study, the frequency of toothbrushing and snacking between meals were the variables that influenced more in root caries.

Effects of two low-shrinkage composites on dental stem cells (viability, cell damaged or apoptosis and mesenchymal markers expression).

To investigate the effects of new two low-shrinkage composites SDR(®) and Venus(®)Bulk Fill on the cell viability, cellular damage and expression of mesenchymal markers on dental stem cells. Specimens from two low-shrinkage composites were eluted with culture medium for 24 h. After 24 h of incubation, cytotoxicity of elutes were evaluated by MTT assay; apoptosis was determined using the DNA-specific fluorochrome Hoechst 33342 and the mesenchymal stem cells markers expression was analyzed by immunofluorescence staining. After 24 h of cell exposure to each extract media, dental stem cells expressed MSCs markers. The interaction among the material and cell line was not significantly correlated [F(1,60) = 2.251, P = 0.39], whereas statistically significant differences among cells lines were observed [F(1,60) = 9.157, P = 0.004], being dental pulp stem cells more resistant that periodontal ligament stem cells. Also, we did not find any significant effect between the tested materials [F(1,60) = 0.090, P = 0.765]. Furthermore, a very low proportion of exposed cells showed condensed or fragmented nuclei, typical of apoptotic cells at 24 h. The results suggest that SDR(®) and Venus(®) Bulk fill and should be considered when selecting an appropriate resin-based dental restorative material.

Heparin enhances protamine disulfide bond reduction during in vitro decondensation of human spermatozoa.

BACKGROUND: Human sperm nuclear decondensation in vivo involves protamine disulfide bond reduction by glutathione (GSH) and protamine/histone exchange, presumably with heparan sulfate (HS) as the protamine acceptor. The aim of the present study was to test the hypothesis that these two events occur simultaneously rather than sequentially, as has been hitherto accepted, and to test for the presence of HS in the human oocyte. METHODS: Spermatozoa and isolated sperm nuclei obtained from normal volunteers were exposed in vitro to heparin, the functional analogue of HS and either GSH or dithiothreitol (DTT) as the disulfide reducing agent. Decondensing reagents were added either simultaneously or sequentially. Percentage sperm nuclear decondensation was assayed by phase contrast microscopy. Thiol reduced status of isolated sperm nuclei was evaluated both indirectly [acridine orange (AO) staining of acid-denatured DNA] and directly [monobromobimane (mBBr) staining of protamine-free thiols]. The presence of HS in mature metaphase II (MII) human oocytes was analyzed by immunocytochemistry. RESULTS: Sequential addition of reagents always resulted in significantly lower decondensation if GSH was used as the disulfide bond reducer (P < 0.05 for sperm and P < 0.001 for nuclei), but only when heparin was used first, when DTT was the disulfide reducing agent (P < 0.05 for sperm and P < 0.01 for nuclei). Both AO staining of DNA and mBBr staining of protamines revealed that the addition of heparin to GSH but not to DTT significantly increased the thiol reduced status of sperm chromatin. HS was detected in the ooplasm of zona-free MII human oocytes. CONCLUSIONS: The results presented in this paper clearly show that heparin enhances the sperm chromatin thiol reducing activity of GSH in vitro, suggesting that in vivo thiol reduction and protamine/histone exchange could occur as simultaneous, rather than sequential, events. We also demonstrate for the first time the presence of HS in the human oocyte.

The presence of heparan sulfate in the mammalian oocyte provides a clue to human sperm nuclear decondensation in vivo.

BACKGROUND: Previous results from our laboratory have led us to propose heparan sulfate (HS) as a putative protamine acceptor during human sperm decondensation in vivo. The aim of this paper was to investigate the presence of glycosaminoglycans in the mammalian oocyte in an effort to better support this contention. METHODS: Two experimental approaches are used: oocyte labeling to identify the presence of HS and analysis of sperm decondensing ability of fresh oocytes in the presence or absence of specific glycosidases. RESULTS: Staining of mouse zona-intact oocytes with the fluorescent cationic dye, Rubipy, at pH 1.5 allowed for the detection of sulfate residues in the ooplasm by confocal microscopy. HS was detected in the ooplasm by immunocytochemistry. A sperm decondensation microassay using heparin and glutathione was successfully developed. The same level of sperm decondensation could be attained when heparin was replaced by mouse zona-free oocytes. Addition of heparinase to the oocyte/glutathione mixture significantly reduced sperm decondensation (P = 0.0159), while there was no effect following addition of either chondroitinase ABC or hyaluronidase. CONCLUSIONS: The results presented in this paper demonstrate for the first time that HS is present in the mammalian oocyte and show that HS is necessary for fresh oocytes to express their sperm decondensing ability in vitro.

In vitro effects of endosulfan-based insecticides on mammalian sperm.

Endosulfan is an organochloride insecticide extensively used in several countries to protect crops from pests. As several studies indicate that endosulfan can affect human and animal development, the aim of this study was to analyse whether sperm parameters and the process of chromatin decondensation could be altered by endosulfan in mice sperm. Spermatozoa from cauda epididymis were obtained from mature male mice and incubated in the presence of two commercial formulations (CFs) of endosulfan (Master® and Zebra Ciagro®) or the active ingredient (AI) alone. A significant decrease in the percentage motility and viability of spermatozoa with respect to controls was found. In vitro decondensation was performed in the presence of glutathione and heparin. Spermatozoa incubated with the AI, endosulfan Master® and endosulfan Zebra Ciagro® showed an increase in chromatin decondensation. In addition, the TUNEL assay showed that DNA fragmentation was significantly higher when sperm were incubated with either one of the CFs when compared to the AI or controls. The ultrastructure analysis of sperm cells showed evident changes in the structure of the plasma and acrosome membranes of sperm incubated with endosulfan AI or the CFs. These results suggest that endosulfan can affect sperm integrity and in vitro chromatin decondensation as well as DNA fragmentation.

Interleukin 6 reduces lipoprotein lipase activity in adipose tissue of mice in vivo and in 3T3-L1 adipocytes: a possible role for interleukin 6 in cancer cachexia.

To investigate whether interleukin 6 (IL-6) might be a potential mediator of the depleted fat reserves observed in malignancy-associated cachexia, we measured lipoprotein lipase (LPL) activity in adipose tissue of mice after administration of IL-6 or tumor necrosis factor and in cultured adipocytes after addition of these cytokines. Injection of IL-6 i.p. reduced adipose tissue LPL activity by 53% within 4.5 to 5.5 h. Injection of tumor necrosis factor elevated serum IL-6 levels and reduced adipose tissue LPL activity by 70%. Both human and murine IL-6 reduced heparin-releasable LPL activity in 3T3-L1 adipocytes in a dose-dependent manner; half-maximal inhibition of LPL activity was achieved with 5000 hybridoma growth factor units/ml. Thus, IL-6 reduces adipose LPL activity and may contribute to the loss of body fat stores associated with some cases of cancer cachexia. Since tumor necrosis factor increases circulating IL-6, some of its effects may be mediated or potentiated by IL-6.

N-acetylcysteine inhibits lipid accumulation in mouse embryonic adipocytes.

Oxidative stress plays critical roles in the pathogenesis of diabetes, hypertension, and atherosclerosis; some authors reported that fat accumulation correlates to systemic oxidative stress in human and mice, but cellular redox environment effect on lipid accumulation is still unclear. In our laboratory we used mouse embryonic fibroblasts (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC) and accumulate lipids, as obesity model. Here we analyzed the role of the well-known antioxidant and glutathione precursor N-acetylcysteine (NAC) in cellular MAPK modulation and lipid accumulation. We evaluated the effect of NAC on the adipogenic differentiation pathway using different doses: 0.01, 0.1, 1 and 5mM; no toxic doses in these cells. A dose of 5mM NAC [DCN-5] provoked a significant decrease in triglyceride accumulation (72±10 [DCN-5] vs 169±15 [DC], p<0.01), as well in Oil Red O stained neutral lipid content (120±2 [DCN-5] vs 139±12 [DC], p<0.01). Molecular mechanisms responsible for adipogenic differentiation involve increase of the expression of phosphoERK½ and phosphoJNK, 5mM NAC treatment inhibited both pERK½ and pJNK protein levels. We also evaluated the mitotic clonal expansion (MCE) which takes place during adipogenesis and observed an increase in DC at a rate of 1.5 cells number compared to CC at day 2, whereas the highest doses of NAC significantly inhibited MCE. Our results suggest that NAC inhibits lipid accumulation and the MAPK phosphorylation in mouse embryonic fibroblasts during adipogenic differentiation and further contribute to probe the importance of cellular redox environment in adipogenesis.

Heparin increases the adhesion of murine mammary adenocarcinoma cells (LM3). Correlation with the presence of heparin receptors on cell surface.

Mastocytosis is a common feature around solid tumors. Due to mast cell (MC) degranulation, heparin and other chemical mediators are released to surrounding tissues. The aim of this paper is to investigate the role of heparin and chemically modified heparins, on a murine mammary adenocarcinoma cell line adhesion properties, and the relationship with the presence of heparin binding sites in tumor cells. We show that heparin increases tumor cell adhesion in a dose-dependent manner. When the number of heparin binding sites was regulated, by culturing the cells with different FCS concentration for 24 hours, a correlation between binding capacity and heparin effect on cell adhesion was observed. The increment on cell adhesion by heparin was lower on cells with less heparin binding sites. Moreover, only heparin and a chemically modified heparin (partially N-desulfated N-acetylated), which bound to heparin-receptor, retained the ability to stimulate cell adhesion, while other modified heparins lost both effects. The increase in cell adhesion was observed on plastic dishes, albumin, as well as on fibronectin pre-coated ones suggesting that heparin effect is substratum independent. Our results show a direct relation between heparin binding to specific cell receptors and increase in cell attachment.

GAL3ST2 from mammary gland epithelial cells affects differentiation of 3T3-L1 preadipocytes.

INTRODUCTION: In the mammary gland, the involution that occurs when lactation ends is an important period for cancer development. We have previously demonstrated stromal-epithelium interactions evaluating conditioned medium of adipose tissue on breast epithelial metalloproteases activity (Creydt et al., Clin Transl Oncol 15:124-131, 2013). Here, we evaluated the effects of conditioned medium of breast epithelial mammary cells on stromal cells. MATERIALS AND METHODS: Conditioned medium from normal murine mammary gland cell line (NMuMG) and conditioned medium proteins were obtained. Then, they were evaluated on modulation of adipocyte differentiation, using 3T3-L1 cell line. RESULTS: We described, for the first time, that breast epithelial mammary cells could produce the enzyme galactose 3-O-sulfotransferase 2 (GAL3ST2). Importantly, GAL3ST2 is present in NMMuMG and two human breast cancer cell lines, and it is more strongly expressed in more metastatic tumors. When 3T3-L1 preadipocyte differentiation was triggered in the presence of conditioned medium from NMuMG or GAL3ST2, triglyceride accumulation was decreased by 40 % and C/EBPß expression by 80 % in adipocytes. In addition, the expression of FABP4 (aP2), another marker of adipocyte differentiation, was inhibited by 40 % in GAL3ST2-treated cells. CONCLUSIONS: Taken together, these results suggest that GAL3ST2 would interfere with normal differentiation of 3T3-L1 preadipocytes; raising the possibility that it may affect normal differentiation of stromal preadipocytes and be a link to tumor metastatic capacity.

Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.

Specific prolactin binding in the rat adrenal gland: its characterization and hormonal regulation.

Rat adrenal prolactin receptors possess the same hormonal specificity as those in the prostate gland and liver, but are less stable during storage and after freezing. There is a gradual decrease in specific prolactin binding to the adrenal during sexual maturation in male rats; maximum binding capacity of 980 fmol/mg protein is at 25 days of age decreasing to approximately 100 fmol/mg protein at day 90. Prolactin receptors in the prostate are high at 25 days of age (700 fmol/mg protein), decrease sharply by day 30 (180 fmol/mg protein) and then gradually increase. Ovariectomy resulted in a significant rise in total prolactin binding in the adrenal gland, while the administration of oestradiol or testosterone reduced the binding, the reverse of changes in prolactin binding in the liver. Only oestrogen increased serum levels of prolactin in female rats. Ovine prolactin (500 micrograms) given to female rats resulted in a rapid increase over a period of 2-8 h total prolactin receptors in the adrenal, and these then decreased to normal levels, indicating a possible positive regulation of prolactin receptors by homologous hormone.

DNA topoisomerase I from Leydig cells is modulated by luteinizing hormone and cyclic adenosine monophosphate.

The action of luteinizing hormone on topoisomerase I activity from rat Leydig cells was studied. Stimulation of the enzyme was observed after long-term (24 and 48 h) gonadotrophin treatment in in vivo experiments. No change could be detected for shorter times than 12 h using two different experimental approaches. Topoisomerase I was stimulated by cAMP in a whole cell extract in a phosphorylation-dependent manner. These results suggest that topoisomerase I could be a target for nuclear events induced by peptide hormone action.

Mitochondrial biosynthesis of cholesterol in Leydig cells from rat testis.

The subcellular location of some enzymes responsible for cholesterol biosynthesis was studied in metrizamide-purified rat Leydig cells. The highest activity of 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMG-CoA reductase), a key regulatory enzyme in the cholesterol pathway, was associated with highly enriched mitochondrial fractions with recovery of 62% of the total activity and was located on the inner membrane. A significant part of the activity (35%) was also present in the cytoplasm. The activity of this enzyme in the other subcellular fractions was negligible. The HMG-CoA synthase activity was also found almost entirely in the mitochondria (90%). Otherwise no detectable activity of HMG-CoA lyase was present in the subcellular fractions studied. Furthermore, cholesterol may be synthesized from acetyl-CoA inside the mitochondrion, since a significant incorporation (90%) of [14C]acetyl-CoA into digitonin-precipitable sterols was observed in this organelle and only 10% in the cytoplasmic fraction. The evidence strongly suggests that much of the cholesterol biosynthesis that takes place in Leydig cells is carried out within the mitochondria.

Effect of a second injection of human chorionic gonadotropin on the desensitized Leydig cells.

Experimental evidence has demonstrated that multiple doses of LH will increase the steroidogenic capacity of Leydig cells. This work was undertaken with the aim of defining the effect of a second hCG administration on the desensitized state, measuring binding of the gonadotropin and the steroidogenic capacity of rat testes. A single injection of 200 IU hCG induced a sharp increase of plasma testosterone which was still evident 24 h later. A second peak was observed at 72 h. The in vivo refractoriness of Leydig cells between 24 and 72 h after the single injection was proved by the fact that a second administration of hCG, 2 h before sacrifice, did not induce any increase in plasma testosterone. A second administration of 200 IU hCG, 48 h after the first injection, showed a similar pattern but on the 5th day there was an increased stimulation of testosterone production with respect to that obtained after a single dose of hCG. The in vitro studies on testicular binding capacity and steroidogenic responsiveness showed that the second administration of hCG, 48 h after the first injection, maintained the testicular binding capacity at the lowest level and the 'adenylate cyclase desensitization' but restored the steroidogenic capacity to even supramaximal values, compared to normal rats, 3 days after this second hCG administration. These results would support a dissociation between receptor loss and maximal testosterone synthesis as well as possibly indicating an alternative pathway different from the classical.

Kinetic properties of microsomal 3beta hydroxysteroid dehydrogenase-isomerase from the testis of Bufo arenarum H.

3beta-hydroxysteroid dehydrogenase 5-ene isomerase (3betaHSD/I) activity is necessary for the biosynthesis of hormonally active steroids. A dual distribution of the enzyme was described in toad testes. The present study demonstrates that in testicular tissue of Bufo arenarum H., microsomal 3betaHSD/I has more affinity for dehydroepiandrosterone (DHEA) than for pregnenolone (K(m)=0.17+/-0. 03 and 1.02 microM, respectively). The Hill coefficient for the conversion of DHEA and pregnenolone were 1.04 and 1.01, respectively. The inclusion of DHEA in the kinetic analysis of pregnenolone conversion affected V(max) while K(m) was not modified, suggesting a non-competitive inhibition of the conversion of pregnenolone. K(i) was calculated from replot of Dixon's slope for each substrate concentration. K(i) from the intercept and the slope of this replot were similar (0.276+/-0.01 and 0.263+/-0.02 microM) and higher than the K(m) for DHEA. The K(m) and K(i) values suggest the presence of two different binding sites. When pregnenolone was present in the assays with DHEA as substrate, no effect was observed on the V(max) while K(m) values slightly increased with pregnenolone concentration. Consequently, pregnenolone inhibited the transformation of DHEA in a competitive fashion. These studies suggest that, in this species, the microsomal biosyntheses of androgens and progesterone are catalysed by different active sites.

Glucagon-like peptide-1(7-36)amide induces the release of aspartic acid and glutamine by the ventromedial hypothalamus of the conscious rat.

Glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) and its own receptor have been found in the hypothalamus and brain stem of the rat. In an attempt to gain further insight into the role of this peptide in brain functioning we investigated the effects of GLP-1 (7-36)amide on the release of excitatory amino acid neurotransmitters by the ventromedial hypothalamus using an experimental microdialysis approach. GLP-1(7-36)amide produced an immediate increase in the extracellular concentrations of aspartic acid and glutamine, p < 0.01 and p < 0.05, respectively. By contrast, extracellular concentrations of glutamic acid, alanine, threonine, and tyrosine were unaffected. The results of this study show a stimulatory effect of GLP-1(7-36)amide on the release of aspartic acid and glutamine by the ventromedial hypothalamus of the rat.

Activity of renal 11betahydroxysteroid dehydrogenase 2 (11betaHSD2) in stressed animals.

The enzyme 11betaHSD2 protects the non-selective mineralocorticoid receptor from occupation by glucocorticoids in aldosterone target tissues. We studied the effect of stress elicited by intubation with a rubber catheter and administration of 10 ml of 0.45% NaCl (G3), of 10 ml of 200 mM HCl (G4) or intubation alone (G2) on the kinetics of the renal enzyme compared with untreated rats (G1). Microsomes were incubated with increasing masses of 3H corticosterone and 400 microM NAD at pH=7.4 during 5 minutes. Samples were extracted with ethyl acetate and analyzed by TLC. Results for n=4: Vmax for G1, 4.82 +/- 0.67. G2, 10.04 +/- 0.16***. G3, 9.16 +/- 0.74**. G4, 10.19 +/- 0.79*** pmoles/min/mg prot. Km for G1, 22.37 +/- 2.42. G2, 50.72 +/- 7.05*. G3, 55.25 +/- 8.37**. G4, 27.40 +/- 3.20 nM. (***p<0.001, **p<0.01 and *p<0.05 vs G1). All treatments increased Vmax. Intubation alone and gavage with 0.45% NaCl, but not with 200 mM HCl, increased Km. Taking together, the results could reflect a way to prevent occupation of type I receptors by increased levels of circulating glucocorticoids due to stressful situations. This protection seems more efficient under acidotic conditions causing--in addition to an increased Vmax--a low Km for the enzyme.

DATALOAD: a user-friendly program to prepare binding data for the "LIGAND" software.

This paper presents a program for the preparation of binding data to be used with the LIGAND software. This program will analyze saturation and competition/displacement binding data, and prepare all the necessary files that provide the input values for the final processing by the LIGAND package. Features include choices of different units for specific activity of the labeled tracer, and screen editing of the data, and results are provided as an average of replicates or as individual points. The program creates a graphic file to observe the raw data as a B/T vs T graph, prior to the analysis with the LIGAND program. The program saves all the information in a session file. Calculation of the bound/free values for the raw data is also provided.

[Evaluation of the cost of an intensive medicine unit. Relationship between the cost and the severity of the disease]. / Valoración del coste de una unidad de medicina intensiva. Relación del coste con la gravedad del enfermo.

BACKGROUND: The evaluation of the costs of intensive care is a subject of interest at present, due to the high resources required by this area of health care services and the rhythm at which these costs increase. Such an evaluation has rarely been carried out in Spain. The aim of this study was to quantify the cost of medical care to critical patients in an Intensive Care Unit (ICU) in addition to evaluate the relationship between the severity of the disease and the short term result of intensive health care. METHODS: A prospective study was carried out in 1,184 patients admitted (February 1985-February 1986) to the ICU of the Hospital General de Especialidades Virgen de las Nieves in Granada (Spain). Variables collected were the severity of the patient (APACHE II), therapeutic intensity (TISS) received, diagnosis on admission and state on discharge. A detailed and individualized evaluation was performed concerning the costs of hospital stay and treatment in the ICU. RESULTS: The cost per patient per day in the ICU was found to 54,438 pesetas in 1988. A significant association was demonstrated with age, severity, therapeutic intensity and the result of the stay in the unit, being much higher in the patients who died in the ICU, particularly in those in whom the prognosis "a priori" was good. CONCLUSIONS: A significant relation was found between the cost and severity of the disease, with the maximum costs being found in patients in whom survival was expected but who died and vice versa.