From Inpatient Notes to Outpatient Followup: Enhancing the Rhinology Service in a Tertiary Centre through Student Led Projects.

Introduction. Medical students can use systems to help improve the quality of care in a unit. Following the review of care within the ENT department at a tertiary centre a number of quality improvement projects were put in place. Methods. The following interventions were established: (1) creation of an outpatient telephone enquiry clinic, (2) development of a rhinology database, (3) introduction of operative note templates, and (4) construction of electronic discharge summary templates (eDSTs). Discussion and Outcomes. (1) Consultant telephone inquiry clinics were successfully organised and showed high levels of patient satisfaction. (2) A database to collect patient reported outcome measures was piloted within rhinology outpatients; the results suggest that such a database would be simple to introduce and yield benefits for patients and the department. (3) Operative note templates for FESS procedures were implemented with a view to improving the continuity of care onto the ward; these have become well established and further steps to integrate these into routine care are being taken. (4) eDSTs specific to FESS and septorhinoplasty procedures were introduced with a view to increasing completion speed of templates and adherence to Royal College of Physician Guidance.

Hardness corrections for copper are inappropriate for protecting sensitive freshwater biota.

Toxicity testing using a freshwater alga (Chlorella sp.), a bacterium (Erwinnia sp.) and a cladoceran (Ceriodaphnia cf. dubia) exposed to copper in synthetic and natural freshwaters of varying hardness (44-375 mg CaCO3/l), with constant alkalinity, pH and dissolved organic carbon concentration, demonstrated negligible hardness effects in the pH range 6.1-7.8. Therefore, the use of a generic hardness-correction algorithm, developed as part of national water quality guidelines for protecting freshwater biota, is not recommended for assessing the toxicity of copper to these, and other, sensitive freshwater species. Use of the algorithm for these sensitive species will be underprotective because the calculated concentrations of copper in water that cause a toxic effect will be higher.

Assessment of metal toxicity in sediment pore water from Lake Macquarie, Australia.

Recent investigations into the level of heavy metal enrichment in the sediments of Lake Macquarie have indicated that significant contamination has occurred over the past 100 years, with elevated levels of lead, zinc, cadmium, copper, and selenium being observed in most parts of the lake. Pore water extracted from sediments showing the greatest contamination by these metals exhibited toxicity to the larval development of the sea urchin Heliocidaris tuberculata. However, an analysis of pore water metal concentrations revealed that the concentrations of these metals were too low to cause toxicity. Rather, pore water toxicity was highly correlated with manganese for the majority of sites sampled; subsequent spiking experiments confirmed manganese as a cause of toxicity. Current levels of manganese in the sediments of Lake Macquarie have arisen from natural sources and are not the result of anthropogenic activities. These results reiterate the importance of identifying the causes of toxicity in assessments of sediment contamination, particularly when testing sediment pore waters using sensitive early life stages.

Fate and toxicity of endosulfan in Namoi River water and bottom sediment.

Endosulfan (6,7,8,9,10,10,-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxathiepine-3-oxide) sorption (standardized to 1% total organic carbon and dry weight) was significantly (P < 0.05) more concentrated on the large (>63 microm) particle fraction compared with smaller size fractions (<5 microm and 5-24 microm) of bottom sediments from the Namoi River, Australia. Following completion of the particle size fractionation (6 to 12 wk) and a sediment toxicity assessment (2 wk), the sediments showed large decreases in concentrations of alpha-endosulfan that coincided with an increase in endosulfan sulfate concentrations and minimal changes in beta-endosulfan concentrations. In the Namoi River, similar patterns were observed in the composition of total endosulfan in monthly measurements of bottom sediments and in passive samplers placed in the water column following runoff from cotton (Gossypium hirsutum L.) fields. The toxicity of endosulfan sulfate in river water indicated by the nymphs of the epibenthic mayfly Jappa kutera, was more persistent than the alpha- and beta-endosulfan parent isomers due to its longer half-life. This suggests that endosulfan sulfate would contribute most to previously observed changes in population densities of aquatic biota. Measured concentrations of total endosulfan in river water of up to 4 microg L(-1) following storm runoff, exceed the range of the 96-h median lethal concentration (LC50) values in river water for both alpha-endosulfan (LC50 = 0.7 microg L(-1); 95% confidence interval [CI] = 0.5 to 1.1) and endosulfan sulfate (LC50 = 1.2 microg L(-1); 95% CI = 0.4 to 3.3). In contrast, the 10-d LC50 value for total endosulfan in the sediment toxicity test (LC50 = 162 microg kg(-1); 95% CI = 120 to 218 microg kg(-1)) was more than threefold higher than the highest measured concentration of total endosulfan in field samples of bottom sediment (48 microg kg(-1)). This suggests that pulse exposures of endosulfan in the water column following storm runoff may be more acutely toxic to riverine biota than in contaminated bottom sediment.

Effect of endosulfan runoff from cotton fields on macroinvertebrates in the Namoi river.

Of the several pesticides used in the pest management strategy for cotton, endosulfan is ranked as having the greatest impact on the riverine ecosystem. A survey of changes in the densities of six abundant macroinvertebrate taxa (ephemeropteran nymphs Jappa kutera, Atalophlebia australis, Tasmanocoenis sp., and Baetis sp. and two trichopteran larvae, Cheumatopsyche sp. and Ecnomus sp.) between upstream and downstream zones of the cotton-growing region in the Namoi River was conducted between November 1995 and February 1996. In November and December 1995, there were few differences in population densities between all sites. In January and February 1996, population densities of the study taxa increased 7- to 10-fold higher at the two reference sites, with low concentrations of endosulfan in sediment and in passive samplers placed in the water column. In contrast, densities of these taxa at sites with exposure to 25-fold higher concentrations of endosulfan remained static and were between one and two orders of magnitude lower than densities at the reference sites in January and February. Population densities of Baetis sp., a mobile ephemeropteran, did not indicate any inverse relationship with endosulfan concentrations. Multivariate redundancy analysis indicated that endosulfan concentrations were the leading environmental predictor of changes in density of the five benethic taxa. Laboratory 48-h LC50 values of technical endosulfan in river water were 0.6, 1.3, and 0.4 ppb for early-instar nymphs of A. australis and J. kutera, and larvae of Cheumatopsyche sp., respectively. Endosulfan sulfate formed a large proportion of the total endosulfan concentrations measured from in situ passive samplers, indicating that its main route of entry into the river is through surface runoff during storm events.

Application of a benthic euryhaline amphipod, Corophium sp., as a sediment toxicity testing organism for both freshwater and estuarine systems.

The use of an as-yet-undescribed euryhaline Corophium sp. amphipod as a sediment toxicity testing organism was assessed. The species was found to be ubiquitous in many tidal areas of the Hawkesbury River catchment. The salinity of habitat sites ranged from 0.1 to 24 ppt, sediment total organic carbon (TOC) ranged from 0.4% to 3.5%, and the fines content (< 63 micron particle size) of the sediment ranged from 4.3% to 47.6%. Monitored populations ranged from a density of 59 to 6622 individuals per m2, with freshwater sites with a sediment fines content greater than 20% having the highest population densities. The sensitivity of the Corophium sp. was assessed by using copper chloride and ammonium chloride as reference toxicants in a 96-h static water-only test and a 10-day static sediment test. The LC50 for copper in freshwater-only exposures was 80 to 86 microg/L, using adult animals collected from the field. In contrast, the LC50 for copper in freshwater sediment and the sediment pore water were 840 mg/kg (dry weight) and 99 microg/L, respectively. The LC50 for ammonia (total) in freshwater-only at pH 7 was 5.5 mg/L. In contrast, the LC50 for ammonia (total) in freshwater sediment and the sediment pore water were 110 mg/kg (dry weight) and 6 mg/L, respectively. Laboratory cultures of 5 per thousand to 15 per thousand salinity were optimal for supporting the release of juveniles. Juveniles collected from laboratory cultures had a LC50 for copper in 5 per thousand and 10 per thousand salinity of 9 microg/L and 28.5 microg/L, respectively, in water-only exposures. The juveniles would be suitable for use in the development of a chronic sediment toxicity test with growth as the endpoint.

Toxicity of acid-sulfate soil leachate and aluminum to embryos of the Sydney Rock oyster.

The toxicity of leachate water from acid-sulfate soil to the early embryonic development of the Sydney Rock oyster, Saccostrea commercialis, was assessed. Concentrations of acid-sulfate soil leachate water as low as 3.3% in seawater were found to decrease the normal development of oyster embryos after 48 hr exposure, and this effect could not be attributed to any significant change in pH or salinity. An EC50 value for the acid-sulfate soil leachate water of 2.5 to 2.9% in seawater was obtained, and the no observed effect concentration was determined at a concentration of 2% in seawater. In tests conducted with aluminum added to seawater, a significant decrease in the percentage of embryos developed to the D-veliger stage occurred at concentrations of 150 micrograms/liter and greater, with no effects at 100 micrograms/liter. An EC50 of 225 micrograms/liter for the effect of added aluminum on embryo survival was obtained and all embryos showed developmental abnormalities at concentrations of 400 micrograms/liter and greater. A significant decrease in the embryonic development occurred when the fertilized eggs were incubated in pH-adjusted seawater at pH values < or = 6.75, but no significant effects were found at pH 7.0 or above. Since aluminum was present in high concentrations in the acid-sulfate soil leachate water, it was concluded that aluminum was the main toxicant in the acid-sulfate water that disrupted the oyster embryonic development.

Toxicity of acid-sulphate soil leachate and aluminium to the embryos and larvae of Australian bass (Macquaria novemaculeata) in estuarine water.

The toxicity of leachate water from acid-sulphate soil to the early life stages of Australian bass, Macquaria novemaculeata, incubated in seawater was evaluated. Acid-sulphate soil leachate water (pH> or =6.8) delayed the hatching of fertilised eggs, but after 48 h the per cent hatching was normal. In comparison, acidic saline water (25 per thousand salinity) at pH 4.0 or less prevented embryos from hatching. The survival of yolk-sac larvae exposed to acid-sulphate soil leachate water at a concentration of 32% in seawater and an initial pH of 7.2, was significantly different to controls after 96 hours. In corresponding tests with only acidified saline water (20 per thousand salinity), pH levels equal to or below 5.0 killed yolk-sac larvae after 96 h exposure. Aluminum showed a pH dependent toxicity to yolk-sac larvae, with added aluminium as low as 200 microg litre(-1) having a significant effect on larval survival at pH 5.5, and concentrations of 600-800 microg litre(-1) having a significant effect on larval survival at an initial pH range of 6.0 < pH < 6.8. It was concluded that significant mortality of the early life stages of Australian bass would occur if they are exposed to acid-sulphate soil leachate that results in a pH in the receiving estuarine water below 5.5, or when the pH is below 6.8 and aluminium is present at a total concentration of 800 microg litre(-1) or greater.

Assessment of the human sperm acrosome reaction using concanavalin A lectin.

A method for assessment of the human sperm acrosome reaction is reported using fluorescein isothiocyanate (FITC)-conjugated Concanavalin A (ConA). The technique involved labelling prefixed spermatozoa, where only those spermatozoa that showed a complete loss of the acrosome bound FITC-ConA to the acrosomal region. Competitive sugar binding studies demonstrated that binding of ConA lectin to the acrosomal area of human spermatozoa was inhibited in the presence of 0.2 M D-mannose. Staining with the supravital stain Hoechst 33258 (H258) concomitantly with FITC-ConA allowed determination of only those spermatozoa that had undergone a true and not degenerative acrosomal loss. Incubation of human spermatozoa with 0, 1, 5, and 25 microM calcium ionophore, A23187, for 60 min demonstrated that changes in acrosomal status due to the different treatment protocols may be determined by the dual-staining method. Electron microscopy studies revealed that gold-conjugated ConA bound specifically to the surface of the inner acrosomal membrane of acrosome-reacted spermatozoa. A significant correlation (r = +.97) between transmission electron microscopy (TEM) and FITC-ConA labelling methods of acrosomal status assessment was achieved. The simple ConA labelling procedure reported here therefore provides a reliable method for quantitation of the physiological acrosome reaction of a population of human spermatozoa.

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro.

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl2 and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl2 revealed that plasma membrane was removed only from the acrosomal region and remained predominantly intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl2 lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg2+, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca2+ (5 mM) to the capacitated spermatozoa induced approximately 78 +/- 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 +/- 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg2+, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.

Sperm antibodies and human in vitro fertilization.

In order to directly evaluate the effects of sperm antibodies in human in vitro fertilization (IVF), the authors preincubated donor sperm in female sera containing sperm antibodies and then inseminated supernumerary human oocytes from a gamete intrafallopian transfer (GIFT) program. The sperm were incubated for 30 minutes in medium containing 20% serum with antisperm activity (Test); or no antisperm activity (Control) as assessed by the immunobead test (IBT). Each oocyte was inseminated with 1 to 2 X 10(5)/ml of the preincubated motile sperm with Control or Test treatments allocated on a random basis. Six positive sera were tested in 17 experiments, resulting in a fertilization rate of 41% (25/61) versus 84% (36/43) for controls (P less than 0.001). When considered individually, three of six positive sera caused significant inhibition. The only serum that gave complete inhibition had the highest titer for IgG (10,000) and lower IgA (100). Absorption with protein A reduced the IgG titer to less than 10 and removed the fertilization inhibitory activity. These results confirm that sperm antibodies from female sera can inhibit human IVF.

Evaluation of Nycodenz and Percoll density gradients for the selection of motile human spermatozoa.

Two high-density media, Percoll (Pharmacia, Uppsala, Sweden) and Nycodenz (Nyegaard & Co., Oslo, Norway), were assessed for efficacy of selection of motile spermatozoa from oligozoospermic and asthenozoospermic semen samples. The results indicated initially that the media were equivalent for motile sperm selection. However, only the spermatozoa selected from discontinuous Nycodenz gradients retained their motility for at least 21 hours (60% motility compared with 5% motility). A significantly higher number of motile spermatozoa were harvested from discontinuous Nycodenz gradients than from matched control samples using the swim-up procedure (12.0 +/- 3.0 standard error of the mean [SEM] X 10(6)/ml versus 1.9 +/- 0.7 SEM X 10(6)/ml; 27 experiments, P less than 0.001). Discontinuous Nycodenz gradients may be useful for selection of motile spermatozoa from poor-quality semen for in vitro fertilization.

Isolation of a stable apical segment of the guinea pig sperm acrosome.

The large apical segments of guinea pig sperm acrosomes were mechanically separated from the spermatozoa and subsequently isolated by density gradient centrifugation. The isolated acrosomal caps were very stable and maintained their crescent morphology when suspended in sucrose-based medium buffered at pH 5.6, with or without the acrosin inhibitor p-aminobenzamidine (pAB). Examination under the electron microscope showed that the acrosomal caps were free of plasma membrane and were bound by an outer acrosomal membrane which was discontinuous. Enzymatic analysis after lysis of the caps indicated that acrosin and hyaluronidase were present with high specific activity, while only a trace amount of acid phosphatase activity and no arylsulphatase, phospholipase A2, or phospholipase C activities were present. Significant particulate acrosin activity, but only trace amounts of soluble acrosin activity, could be detected in the isolated acrosomal caps if assayed immediately after isolation in the absence of pAB. However, soluble acrosin activity of high specific activity was obtained after the acrosomal caps were extracted by 10% glycerol buffered at low pH (pH 3.0). The new procedures provide a means to isolate and purify guinea pig sperm apical acrosomal segments rapidly.

Pregnancy from in vitro fertilization of human eggs after separation of motile spermatozoa by density gradient centrifugation.

The spermatozoa of infertile men, previously found to have poor fertilizing capacity in an in vitro system, were fractionated on discontinuous gradients of Percoll (Pharmacia Laboratories, Piscataway, NJ) and were used for insemination of human oocytes in an in vitro fertilization and embryo replacement program. Three of the five patients in the study had established ongoing pregnancies, including one patient with a triplet pregnancy from four transferred embryos. However, the other two patients continued to have poor fertilizing capacity with their husband's Percoll-separated spermatozoa.

Influence of 2-deoxy-D-glucose and energy substrates on guinea-pig sperm capacitation and acrosome reaction.

When guinea-pig spermatozoa were suspended in a minimal culture medium (MCM-PL), 2-deoxy-D-glucose (100 microM) and 2-amino-2-deoxy-D-glucose (100 microM) were potent inhibitors of the acrosome reaction without affecting the sperm motility, whereas the N-acetyl derivative 2-acetamido-2-deoxy-D-glucose (2 mM) had no inhibitory effect. The addition of D-glucose (2 mM) partly inhibited the percentage acrosome reaction of spermatozoa suspended in Medium MCM-PL, but DL-alpha-glycerophosphate (2 mM) and myo-inositol (2 mM) had no effect. In addition, DL-alpha-glycerophosphate (2 mM) did not overcome the inhibitory effect of 2-deoxy-D-glucose on the sperm acrosome reaction. The inhibitory action of 2-deoxy-D-glucose (100 microM) on the sperm acrosome reaction assessed after a 3-h incubation was irreversible and was only completely effective if the sugar was added within 30 min of the start of incubation. When spermatozoa suspended in Medium MCM-PL were treated with 2-deoxy-D-glucose (100-200 microM) for an extended incubation up to 6 h, the inhibitory effect of 2-deoxy-D-glucose was partly overcome. Spermatozoa treated with 2-deoxy-D-glucose had significantly reduced concentrations of ATP after incubation for 2 h in Ca2+-free media, compared with the ATP concentrations of spermatozoa preincubated for 2 h in Ca2+-free media that supported acrosome reactions. The addition of Ca2+ (5 mM) caused a rapid decrease in ATP concentrations of spermatozoa suspended in Medium MCM-PL, while the addition of the monovalent ionophore monensin (50 microM) and Ca2+ stimulated sperm acrosome reactions as well as an additional decline in the sperm ATP concentrations. However, monensin (50 microM) in the absence of Ca2+ caused only a slight decline in the sperm ATP concentrations over the 15-min incubation period. The depletion of the sperm ATP concentrations by 2-deoxy-D-glucose may retard completion of the capacitation process and the resultant acrosome reaction.

Bicarbonate- and calcium-dependent induction of rapid guinea pig sperm acrosome reactions by monovalent ionophores.

The monovalent cationic ionophores monensin and nigericin stimulated rapid guinea pig sperm acrosome reactions in the presence of extracellular Na+, Ca2+ and bicarbonate (HCO3-/CO2). Extracellular K+ (mM concentrations), in contrast, was not required for the stimulatory effect of the ionophores. The effect of HCO3-/CO2 is concentration, pH and temperature dependent, with maximal responses obtained with 50 microM monensin or 25 microM nigericin at a concentration of 30 mM HCO3-, 2.5% CO2 and pH 7.8 at 25 degrees C. At a constant HCO3- concentration (30 mM), monensin stimulated acrosome reactions within the pH range 7.5-7.8, whereas a higher or lower pH did not support acrosome reactions at 25 degrees C. At constant extracellular pH (7.8), monensin stimulated acrosome reactions in the presence of 30 mM HCO3-, whereas higher and lower concentrations did not support acrosome reactions at 25 degrees C. The permeant anions pyruvate and lactate were essential to maintain sperm motility when treated with monensin under these conditions. NH4Cl, sodium acetate and 4,41-diisothiocyano-2, 21-disulfonic acid stibene (DIDS; 25 microM), an anion transport inhibitor, blocked the ability of monensin to stimulate acrosome reactions. Verapamil (100 microM), a putative Ca2+ transport antagonist, in contrast, did not prevent the monensin-induced acrosome reactions. Physiological concentrations of Na+ were needed for monensin to stimulate acrosome reactions, but high concentrations of Mg2+ prevented the monensin stimulation. The Ca2+ ionophore A23187 (75 nM) also required physiological concentrations of Na+ for the rapid induction of maximal acrosome reactions at an elevated pH (8.3) but did not require the presence of extracellular HCO3-. These studies suggest that a monovalent ionophore-induced rise in sperm intracellular Na+ concentrations is a pre-Ca2+ entry event, that stimulates an endogenous Ca2+/Na+ exchange that allows a Ca2+ influx which in turn induces the acrosome reaction. The possible regulatory role of the sperm intracellular pH and Na+, K+-ATPase during the capacitation process under physiological conditions is discussed.

A technique for enrichment of motile spermatozoa from oligozoospermic and asthenozoospermic patients.

A system for rapid selection of normal motile spermatozoa from poor semen samples is described. Analysis of the selected samples shows that there are significant improvements in the percentage motility, velocity and morphology. The final spermatozoa suspension of 0.5 ml is sufficient for the purpose of intrauterine AIH and IVF.

Sodium requirement for capacitation and membrane fusion during the guinea-pig sperm acrosome reaction.

Guinea-pig spermatozoa, incubated in a minimal culture medium (MCM-PL) containing 150 mM-Na+ and no added K+, capacitate within 2 h and respond to added Ca2+ with maximal percentage of acrosome reactions. When spermatozoa, initially capacitated in Medium MCM-PL, were washed and resuspended in saline-based media, the motile spermatozoa showed acrosome reactions only in response to added Ca2+ at an extracellular pH greater than 7.4. In contrast, resuspension of capacitated spermatozoa into isotonic sucrose or choline-based media containing no added Na+ and K+ (pH 7.8) did not support acrosome reactions in response to the addition of Ca2+. Examination under the electron microscope showed that these spermatozoa responded to added Ca2+ with swelling or cavitation of the acrosomal matrix but fusion of the plasma membrane and acrosomal membrane did not occur. Treatment of spermatozoa with monensin, a monovalent cationic ionophore, induced rapid acrosome reactions in the presence of extracellular Ca2+ and Na+. In contrast, spermatozoa incubated for 3 h in a sucrose-based Na+-deficient medium or Medium MCM-PL containing 10 mM-KCl failed to give a significant percentage of acrosome reactions in response to the addition of Ca2+, but the addition of ouabain (0.1 mM) prevented the K+ inhibition. Amiloride, a sodium channel blocker in various other tissues, retarded the development of acrosome reactions of spermatozoa incubated in Medium MCM-PL. Based on these data, it appears that an increase in intracellular Na+ has a functional role in acquisition of guinea-pig sperm fertilizing ability.

Effects of putative protease inhibitors on the acrosome reaction of sea urchin spermatozoa.

The acrosome reaction was induced by jelly coat factors, nigericin, or elevated pH. When spermatozoa were preincubated for 5 min in sea water maintained at pH 7.9 in the presence of 1 mM-phenylmethylsulphonyl-fluoride (PMSF), 1 mM-benzamidine, 0.1 mM-1-chloro-3-tosyl-amido-7-amino-2-heptanone (TLCK), 5 mM-diisopropylphosphofluoridate (DFP) or 5 mM-DFP that was previously hydrolysed, only DFP or its hydrolysis product(s) prevented formation of the acrosomal filament induced by jelly coat factors. When incubation with inhibitors was extended to 2 h only TLCK and its hydrolysis products inhibited the jelly-induced acrosome reaction. Only DFP significantly inhibited the acrosome reaction induced by elevated pH (9.0). Nigericin induced acrosome reactions in the presence of DFP or TLCK. These findings do not support the concept of an active role for acrosin in development of an acrosome reaction.