Pregnancy and birth after intracytoplasmic sperm injection of in vitro matured germinal-vesicle stage oocytes: case report

OBJECTIVE: To report a normal pregnancy and the delivery of a healthy child after the combination of in vitro maturation of germinal-vesicle stage oocytes and intracytoplasmic sperm injection (ICSI) in a patient. SETTING: Procedures were performed in a tertiary IVF center coupled with an institutional research environment. MAIN OUTCOME MEASURES: Maturation rate of immature oocytes after in vitro maturation and intactness, fertilization, and developmental rates of oocytes after microinjection. RESULTS: Nine of 14 germinal-vesicle stage oocytes matured to the metaphase II stage after 30 hours of in vitro culture (64%). Seven of eight injected and intact oocytes fertilized normally (78%) and five of them cleaved with < 20% fragmentation (71%). Four embryos were transferred and a singleton pregnancy was obtained that ended in the delivery of a healthy child. CONCLUSION: In vitro maturation of immature oocytes together with ICSI can result in normal fertilization, embryo development, pregnancy, and the delivery of healthy child.     

Embryo cloning in cattle: the use of in vitro matured oocytes

In vitro matured (IVM) bovine oocytes were examined to determine their potential viability in embryo cloning. Activation competence, as monitored by pronuclear formation, increased with oocyte age. Oocytes readily formed a pronucleus when challenged with an electrical pulse 30 h after the onset of maturation. Developmental competence of IVM oocytes tended to increase with oocyte age (P = 0.079). Selection of IVM oocytes on the basis of the presence of a polar body 24 h after the onset of maturation and the size of the follicle from which the oocyte was derived improved development of nuclear transfer embryos (polar body positive 25% versus polar body negative 10%, P < 0.05; large follicle oocytes 31% versus small follicle oocytes 14%, P < 0.05). When selected, IVM oocytes were compared with in vivo matured oocytes recovered from superovulated cows and heifers; no difference was detected for the frequency of embryos produced, pregnancies confirmed between days 50 and 60 of gestation, or the number of calves born. We conclude that selected IVM oocytes are equivalent to in vivo matured oocytes when used for bovine embryo cloning.     

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.     

Protein neosynthesis by porcine oocytes matured in vivo and in vitro

The protein pattern of individual porcine oocytes matured as intact cumulus oocyte complexes either in vivo or in vitro with or without FSH and LH for 43 h were investigated. The synthesis of a 53 kDa polypeptide ceased 21 h after hCG administration whereas a 44 kDa polypeptide were consistently absent in the protein patterns of nearly all of the in vivo maturing oocytes. Further on, a polypeptide with a relative molecular weight of 46000 persisted throughout maturation. A precipitous increase in the synthesis of two other proteins with relative molecular weights of 38000 and 28000, respectively, was observed at 9 and 21 h after hCG injection. In in vitro matured oocytes with or without FSH and LH the synthesis of the 53 kDa band decreased after a culture period of 9h. Further on, the production of the 44 kDa polypeptide ceased only in oocytes incubated in FSH and LH supplemented media after 21 h of culture. On the other hand, the two proteins of Mr 38000 and 28000 appeared only in most of the protein profiles of oocytes cultured with FSH and LH for 43 h. The production of the 46 kDa polypeptide during a 21 h culture period was significantly affected by the presence of additional granulosa cells in connection with the cumulus oocyte complex. Neither the appearance nor the disappearance of the 5 investigated bands was influenced by the presence or absence of the germinal vesicle after 21 h of culture. It is concluded that at least the addition of FSH and LH to the culture medium is necessary for cumulus oocytes complexes to synthesize a protein pattern closely corresponding to that produced by in vivo matured oocytes.     

Fertilization of bovine oocytes grown in vitro

Early antral follicles 0.5-0.7 mm in diameter were dissected from bovine ovaries, and oocyte-cumulus complexes with pieces of parietal granulosa (OCCGs) were then collected from the follicles. The OCCGs containing oocytes of 90-99 microm diameter (94.7+/-2.8 microm, n = 196) were selected and embedded in collagen gels and cultured for 14 days in TCM199 containing 10% fetal calf serum and 4 mM hypoxanthine. From cultured OCCGs, 144 surviving oocytes were recovered, of which 53 were granulosa cell-enclosed oocytes and 91 were denuded oocytes. The mean diameter of the surviving oocytes was 114.2+/-8.4 microm, significantly larger than that measured before culture (P < 0.05). The granulosa cell-enclosed oocytes and denuded oocytes were further cultured for maturation for 24 h. After culture, 72% (38/53) of the granulosa cell-enclosed oocytes and 59% (54/91) of the denuded oocytes showed normal morphology. These oocytes were then inseminated with bovine spermatozoa. After 29 h of insemination, all of the denuded oocytes had degenerated, while 32% (12/38) of the granulosa cell-enclosed oocytes showed normal morphology. Of the 12 oocytes, 5 were penetrated by spermatozoa, and 2 formed both male and female pronuclei. These results demonstrate for the first time that bovine oocytes grown in vitro acquire meiotic competence and can be penetrated by spermatozoa.     

Activation of in vitro matured mouse oocytes arrested at first or second meiotic metaphase

Some mammalian oocytes fail to complete maturation in vitro and arrest development at the first metaphase stage. The response of such blocked oocytes to sperm penetration was investigated. Ovarian mouse oocytes from two inbred strains, CBA/Kw and KE, were cultured in vitro for 20 h. Both oocytes arrested at the first metaphase (MI oocytes) and second metaphase (MII oocytes) were then inseminated. The majority of MII and MI oocytes reinitiated meiosis in response to sperm penetration, although those from the CBA strain did with higher frequency. Moreover, a high proportion of unpenetrated oocytes from CBA, but not the KE strain, resumed meiosis (33% for MII and 48% for MI oocytes, respectively). Parthenogenetic activation of MI-arrested oocytes was demonstrated in (CBAxKE)F1 mice; ovarian oocytes matured in vitro and then treated by electric shock were activated with a similar total frequency of 52.4% for MI and 47.8% for MII oocytes. The rate of activation increased equivalently for both MI and MII oocytes as the length of maturation prolonged. This demonstrates that mouse oocytes arrested at MI during their maturation in vitro continue cytoplasmic maturation and become capable of undergoing activation in a way similar to those maturing to MII. Additionally, in MII oocytes cultured for an equal time in vitro the rate of activation increased with the time lapse after first polar body (PB1) extrusion. This indicates that after PB1 extrusion, the oocyte requires some resting time before it may be activated, perhaps to restore the proper balance between elements of the cell cycle controlling the mechanism involved in first meiotic division.     

The correlation of sperm chromatin decondensation following in vitro exposure to heparin and sperm penetration rates

PURPOSE: The aim of this study was to evaluate the possible correlation of low-dose heparin-induced decondensation of sperm chromatin with sperm concentration, motility, morphology, membrane hypoosmotic response, ejaculate volume, and the ability of sperm to penetrate zona-free hamster oocytes. METHODS: Twenty-two donors of known fertility and 105 patients undergoing evaluation at an andrology laboratory were evaluated by standard World Health Organization semen analysis techniques and a modified sperm penetration assay (SPA). An aliquot was also incubated for 60 min and Ham's F10 medium containing 50 USP/ml heparin. The percentage of sperm undergoing chromatin decondensation was evaluated and correlated to SPA rates and semen quality parameters. RESULTS: No significant correlation was observed between semen parameters and decondensation rates. A nonsignificant (P = 0.11) inverse correlation (P = -0.21) was observed between SPA rates and chromatin decondensation. Significant (P < 0.001) differences were observed in the decondensation rate of donors (3.7 +/- 0.6), patients with normal SPA rates (7.8 +/- 1.5), and patients with decreased SPA rates (21.7 +/- 1.8). The decondensation rates were significantly different (P < 0.01) between patients with a normal SPA rate and patients with a decreased SPA rate. CONCLUSIONS: These data indicate a significant inverse relationship between the SPA rate, which has previously been shown to correlate highly with fertilization ability and heparin-induced sperm chromatin decondensation.     

Developmental competence, after transfer to recipients, of porcine oocytes matured, fertilized, and cultured in vitro

The objective of this study was to evaluate the developmental ability of early porcine embryos produced in vitro and transferred to recipient gilts. Porcine cumulus-oocyte complexes were matured in modified North Carolina State University-37 solution for 44-46 h (in vitro maturation, IVM). In vitro fertilization (IVF) was performed with frozen-thawed epididymal spermatozoa. Inseminated oocytes were cultured in vitro (IVC) for 0, 24, or 48 h in modified NCSU-37 solution. Embryos were surgically transferred to the oviducts of recipients in which estrus had been synchronized with eCG and hCG. On the 29th day post-IVF, the uteri of some recipients were surgically examined for pregnancy; then pregnant females were hysterectomized in order to examine number and weight of the fetuses. Developmental rates to fetuses for IVM/IVF oocytes cultured for 24 and 48 h were significantly lower (p < 0.05, 1.7% and 2.0%, respectively) than that of IVM/IVF oocytes without IVC (6.7%). However, the weights of fetuses (1.0-1.2 g) did not differ among the experimental groups. The other recipients were examined for pregnancy using an ultrasound pregnancy detector, and pregnant females were allowed to go to term. Healthy piglets were delivered by some recipients to which embryos cultured for 0 or 24 h had been transferred; however, no farrow was obtained from embryos cultured for 48 h before the transfer. The results indicate that the viability of in vitro-produced porcine embryos is decreased by IVC after IVF; however, these embryos have competence to develop to term. An improved IVC system of porcine IVM/IVF oocytes is needed to generate advances in this field.     

Intracytoplasmic sperm injection of in vitro-matured equine oocytes

Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and processed for light and transmission electron microscopy. Single pronucleus formation was observed in 2 out of 12 presumptive zygotes 10 h postinjection, at which time abundant cortical granules were observed in the subplasmalemmal region. Twenty hours postinjection, however, 2 pronuclei were observed in 6 of 12 injected oocytes (fertilization rate 50%), and almost all cortical granules were released. The cleavage rate in vitro was 16% after 72 h in culture, and the most advanced embryo stages obtained were 6- to 8-cell embryos. The cleavage rate in vivo was very low since only 1 of 10 recovered had cleaved to the 2-cell stage. Thus, in conclusion, ICSI fertilization of equine oocytes did result in fertilization, pronucleus formation, and cortical granule release. However, the observed fertilization rate and oocyte activation was not paralleled by substantial cleavage of the zygotes.     

Dynamics of maturation-promoting factor and its constituent proteins during in vitro maturation of bovine oocytes

Maturation-promoting factor (MPF) is known to be a key regulator of both mitotic and meiotic cell cycles. MPF is a complex of a B cyclin and the cyclin-dependent kinase cdkl (p34cdc2). Oocyte maturation and its arrest at metaphase of meiosis II (MII) are regulated by changes in MPF activity. In this study, experiments were conducted to examine the dynamics of MPF activity and its constituent proteins during in vitro maturation of bovine oocytes. Bovine oocytes displayed relatively low levels of MPF (histone H1 kinase) activity at the germinal vesicle stage during the first 8 h of maturation. MPF activity increased gradually thereafter, and its first peak of activity occurred at 12-14 h of maturation (presumptive metaphase I), which was followed by an abrupt reduction in activity at 16-18 h, during presumptive anaphase and telophase. MPF activity then increased, reaching a plateau at 20-24 h of maturation (MII stage). This high level of MPF activity was maintained for several hours but decreased gradually after 30 h of maturation and became barely detectable by 48 h of in vitro maturation (IVM) culture. At each time point, there was a significant variation among individual oocytes in histone H1 kinase activity, which was probably due to asynchronous maturation. Abundance of cdk1 increased gradually during the first 8 h and then remained relatively constant except for an apparent reduction at 18-22 h of IVM. The level of cyclin B2 increased quickly during the initial 2 h of culture, and this high level was maintained until 16 h, after which a significant reduction was observed between 18 and 22 h of IVM. The de novo synthesis of cyclin B2, however, exhibited a biphasic oscillation during maturation, with peaks before the onset of MI and of MII. These results have defined the profiles of MPF activity and its individual components during bovine oocyte maturation in vitro. We conclude that active MPF regulates bovine oocyte maturation and that de novo synthesis of cyclin B2 occurs during the process of maturation.     

Amino acids in maturation medium and presence of cumulus cells at fertilization promote male pronuclear formation in porcine oocytes matured and penetrated in vitro

The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The addition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro.     

Effect of follicular or oviductal fluids on movement characteristics of bovine sperm during capacitation in vitro

Mammalian sperm exhibit characteristic motility changes associated with capacitation. Movement characteristics of bovine sperm incubated in noncapacitating (control, medium alone), capacitating (oviduct fluid, nonluteal, and luteal), or capacitating, acrosome reaction inducing (follicular fluid) conditions were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4 hours in a modified Tyrode's medium (control), 20 and 60% nonluteal (NL) or luteal (L) oviduct fluid (ODF), or 20 and 60% follicular fluid (FF). Relative to sperm incubated in control medium, motility of sperm treated with ODF or FF had increased linearity and vigorous motility. Sperm incubated in 60% ODF or FF showed a small decrease in mean trajectory/path straightness and velocity over time compared to 20% fluid treatments and control. Frequency distribution graphs were symmetric for 20% NL- and L-ODF treated sperm. However, 20% FF and 60% ODF and FF treatments had distributions skewed to the left, indicating smaller values for lateral head displacement (ALH) and curvilinear velocity (VCL). Median values for ALH and VCL were determined for control-treated sperm, and subtracted from individual sperm values for all treatments to estimate deviation from control, designated ALHc and VCLc. Three-dimensional plots of ALHc, VCLc and corresponding frequency indicated shifts in peak patterns for fluid-treated sperm compared to control sperm. Incubation in 20% ODF and FF resulted in peak shift for ALH and VCL values; yet, little change in peak position was observed in sperm incubated in 60% ODF and FF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energy substrates and amino acids provided during in vitro maturation of bovine oocytes alter acquisition of developmental competence

Clinicians commonly include an assessment of leg length inequality (LLI) as a component of a musculoskeletal examination. Little research is available, however, documenting reliability and validity of clinical methods for assessing LLI. The purpose of this study was to determine the reliability and validity of assessing functional LLI using a pelvic leveling device. Subjects were 19 women and 13 men between the ages of 18 and 55 who reported having a diagnosed or suspected LLI. Clinical determination of LLI was made by placing rigid lifts under the suspected shorter lower extremity until the leveling device indicated that the iliac crests were level. This measurement was made twice by one investigator and once by a second investigator. Standing radiographic measurements of LLI using rigid lifts were used to establish validity of the clinical method. Intraclass correlation coefficients [ICC(2,1)] and absolute difference values were computed to assess reliability and validity. The mean absolute difference between the two clinical measurements of LLI by the same investigator was 0.29 cm (+/- 0.52), with an ICC = 0.84. The mean absolute difference between clinical measurements of LLI by the two investigators was 0.49 cm (+/- 0.46), with an ICC = 0.77. The ICC and mean absolute difference reflecting agreement between radiographic measurements and clinical measurements of LLI was 0.64 and 0.58 cm (+/- 0.58), respectively, for one investigator and 0.76 and 0.55 cm (+/- 0.37), respectively, for the second investigator. The intratester reliability, intertester reliability, and validity assessments included instances in which paired observations disagreed regarding which lower extremity was the shorter lower extremity. Factors that may be associated with the unacceptable reliability and validity of the clinical assessment method include asymmetric positioning of the ilia, body composition of the patient, and design of the clinical instrument. The authors discuss clinical implications related to assessment of LLI.     

Electron microscopic observations on sperm penetration in cytochalasin-treated eggs of the rose bitterling

The influence of Aloe vera (L.) Burman f. on the glycosaminoglycan (GAG) components of the matrix in a healing wound was studied. Wound healing is a dynamic and complex sequence of events of which the major one is the synthesis of extracellular matrix components. The early stage of wound healing is characterized by the laying down of a provisional matrix, which is then followed by the formation of granulation tissue and synthesis of collagen and elastin. The provisional matrix or the ground substance consists of GAGs and proteoglycans (PGs), which are protein GAG conjugates. In the present work, we have studied the influence of Aloe vera on the content of GAG and its types in the granulation tissue of healing wounds. We have also reported the levels of a few enzymes involved in matrix metabolism. The amount of ground substance synthesized was found to be higher in the treated wounds, and in particular, hyaluronic acid and dermatan sulphate levels were increased. The levels of the reported glycohydrolases were elevated on treatment with Aloe vera, indicating increased turnover of the matrix. Both topical and oral treatments with Aloe vera were found to have a positive influence on the synthesis of GAGs and thereby beneficially modulate wound healing.     

Effect of accessory sex gland fluid from bulls of differing fertilities on the ability of cauda epididymal sperm to penetrate zona-free bovine oocytes

The ability of accessory sex gland fluid to affect the fertility of cauda epididymal sperm was evaluated for 10 bulls that ranged in fertility from 6.2% below to 6.0% above the average fertility of bulls at artificial breeding cooperatives. Cauda epididymal sperm collected from indwelling vasa deferentia catheters and cauda epididymal sperm exposed to accessory sex gland fluid from the same bull were compared on the basis of their rates of in vitro penetration of zona-free oocytes after heterospermic insemination. Incubation of cauda epididymal sperm with accessory sex gland fluid significantly enhanced the ability to penetrate oocytes, and bull fertility affected the magnitude of this improvement. For bulls of average and higher fertility, the positive influence of accessory sex gland fluid on penetrating ability of sperm was highly significant (p < 0.0001). Accessory sex gland fluid from bulls of below-average fertility also improved the penetrating ability of cauda epididymal sperm, although not significantly (p = 0.07). Heterospermic competitions compared the penetrating ability of cauda epididymal sperm exposed to homologous accessory sex gland fluid with a portion of the same sperm population incubated in heterologous accessory sex gland fluid from a bull of contrasting fertility. In experiments involving sperm from 12 different bulls, paired in 42 fertile/subfertile combinations, samples of cauda epididymal sperm mixed with accessory sex gland fluid from the higher-fertility bulls had greater oocyte-penetrating ability than when aliquots of that sample were mixed with accessory gland fluid from lower-fertility bulls (p < 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of multiple centrioles in a rhesus macaque oocyte matured in vitro

This report demonstrates for the first time the presence of centrioles in a rhesus macaque oocyte which had attained meiotic maturation in culture. The exceptional presence of centrioles in this oocyte was associated with anomalous patterns of chromosomal and spindle organization.     

Decondensation of sperm nuclei in vitro

The urine of five patients with three distinct diseases ("I Cell disease" and two new types of mucolipidosis) contains sialic acid-rich oligosaccharides in a high amount: 50- to 500-fold the normal. The structure of the major components are as follows: alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 3)betaMan(1 leads to 4)GlcNac,[alphaAcNeu(2 leads to 6)]betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[betaGal(1 leads to 4)betaGlcNac(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc and alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 3)[alphaAcNeu(2 leads to 6)betaGal(1 leads to 4)betaGlcNAc(1 leads to 2)alphaMan(1 leads to 6)]betaMan(1 leads to 4)GlcNAc. These results suggest that a deficit in alpha-neuraminidase is associated to these three different disorders and that an endo-beta-D-N-acetylglucosaminidase is able to release sialyoligosaccharides by splitting the sialylglycans of glycoproteins.     

Influence of formulation on the in vitro transdermal penetration of flutrimazole

Flutrimazole (1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1 H-imidazole, CAS 119006-77-8, UR-4056) is a new wide spectrum local imidazolic antifungal agent that has already been formulated as a dermal cream (FDC). A comparative study was carried out of the release of flutrimazole from two emulsions in which the drug has been incorporated differently: one dissolved in the oily phase (E24) and the other dispersed in the aqueous formulation phase (E25). Based on the E25 formulation, two more dermal creams were prepared, E27 with benzyl alcohol and E28 with diazolidinyl urea as preservative agents. A comparative study of transdermal penetration including E27, E28, FDC (reference 1% flutrimazole dermal cream) and 1% flutrimazole hydroalcoholic solution was also performed. An amount of the sample dosage form containing 10 mg of flutrimazole was applied to a Franz type cell. The penetration membrane used was cellulose acetate in the release studies and human skin provided by a plastic surgery clinic in the transdermal penetration study. The amount released after 7 h was 36.3 +/- 4.9 micrograms when flutrimazole was dissolved (E24) and 35.9 +/- 5.3 micrograms when flutrimazole was dispersed (E25). Although the differences were not significant, the cream with dispersed flutrimazole was selected for further penetration studies due to its better stability observed in previous studies. The amounts of drug penetrated after 44 h were 31.3, 41.5, 38.3 and 186.5 micrograms for E27, E28, FDC dermal creams and topical hydroalcoholic solution, respectively. The solution showed a statistically significant difference (p < 0.05) from the other formulations, however, no differences were observed between the dermal cream formulations. No differences were neither obtained between the different dermal creams when the amount of drug retained in the skin was compared. This allows to assert that the excipients used do not have different influences on transdermal penetration. In all cases, the mean quantity penetrated in relation to the dose applied was at most 0.5%. These results allow to infer that flutrimazole shows scarce transdermal penetration. Further, the amount of flutrimazole retained per gram of skin is more than 100 times the MIC per gram obtained in previous in vitro studies. It may be assumed that the topical application of the new formulations assayed would allow to obtain a good therapeutic response.