Anti-Mullerian hormone-tailored stimulation protocols improve outcomes whilst reducing adverse effects and costs of IVF.

BACKGROUND: Anti-Müllerian hormone (AMH) is increasingly used to quantify ovarian reserve, but it has not yet realized its full clinical potential in assisted reproduction technology. We investigated the possible benefits of using novel, stratified ovarian hyperstimulation protocols, tailored to individual AMH levels, compared with conventional stimulation. METHODS: Retrospective data were collected from 769 women (first cycle of IVF, using fresh embryos), in a UK tertiary care unit: 346 women using conventional stimulation protocols; 423 women treated under new AMH-tailored protocols. RESULTS: Embryo transfer rates increased significantly (79-87%: P= 0.002) after the introduction of AMH-tailored stimulation protocols. Pregnancy rate per cycle started and live birth rate also increased significantly compared with conventionally treated women (17.9-27.7%, P= 0.002 and 15.9-23.9%, P = 0.007, respectively). Moreover, in the AMH group, the incidence of the ovarian hyperstimulation syndrome (OHSS) fell significantly (6.9-2.3%, P = 0.002) and failed fertilization fell from 7.8 to 4.5%. The cost of fertility drug treatment fell by 29% per patient and the overall cost of clinical management of OHSS fell by 43% in the AMH group. GnRH antagonist protocols, introduced as part of AMH-tailored treatment, may have contributed to the observed improvements: however, within the AMH-tailored group, the live birth rate was not significantly different between agonist and antagonist-treated groups. CONCLUSIONS: Although large, prospective, multicentre studies are indicated, we have clearly demonstrated that individualized, AMH-guided, controlled ovarian hyperstimulation protocols significantly improved positive clinical outcomes, reduced the incidence of complications and reduced the financial burden associated with assisted reproduction.

Antioxidant properties of colchicine in acute carbon tetrachloride induced rat liver injury and its role in the resolution of established cirrhosis.

Antioxidant and antifibrotic properties of colchicine were investigated in the carbon tetrachloride (CCl(4)) rat model. (1) The protective effect of colchicine pretreatment on CCl(4) induced oxidant stress was examined in rats subsequently receiving a single lethal dose of CCl(4). Urinary 8-isoprostane, kidney and liver malondialdehyde and kidney glutathione levels increased following CCl(4) treatment, but only the rise in kidney malondialdehyde was significantly inhibited by colchicine pretreatment. Serum total antioxidant levels were significantly higher in the colchicine pretreatment group. (2) The long term effects of colchicine treatment on CCl(4) induced liver damage were investigated using liver histology and biochemical markers (hydroxyproline and type III procollagen peptide). Co-administration of colchicine with sub-lethal doses of CCl(4) over 10 weeks did not prevent progression to cirrhosis. However, rats made cirrhotic with repeated CCl(4) challenge and subsequently treated with colchicine for 12 months, all showed histological regression of cirrhosis. (3) The antioxidant effect of colchicine in vitro was evident only at very high concentrations compared to other plasma antioxidants. In summary, colchicine has only weak antioxidant properties, but does afford some protection against oxidative stress; more importantly, long term treatment with this drug may be of value in producing regression of established cirrhosis.

Gluten-sensitive enteropathy in Irish setter dogs: characterisation of jejunal microvillar membrane proteins by two-dimensional electrophoresis.

This study investigated whether gluten-sensitive enteropathy (GSE) in Irish setter dogs was associated with underlying structural abnormalities of microvillar membrane proteins. Jejunal biopsies taken from eight-month-old GSE-affected dogs reared on a normal, gluten-containing diet exhibited partial villous atrophy and contained more intra-epithelial lymphocytes than controls. The morphological abnormalities were reversed by feeding a gluten-free diet for five months and the changes were accompanied by an increase in the mucosal activity of the microvillar hydrolases, particularly aminopeptidase N and dipeptidyl aminopeptidase IV, which reverted to pre-treatment levels after a gluten challenge. Two-dimensional electrophoresis of microvillar membrane proteins isolated from GSE-affected dogs revealed an essentially normal protein map that was comparable to controls. The exception was an intense 85 kDa protein spot that diminished when the affected dogs were fed a gluten-free diet and re-intensified after a gluten challenge.

An aminopeptidase N deficiency in dog small intestine.

This study has identified a naturally occurring, specific deficiency of a brush border aminopeptidase N (ApN) in the small intestines of five clinically healthy dogs. ApN activity in mucosal homogenates of dog small intestine was reduced significantly in deficient animals (13.4 (1.1) nmol min-1 mg-1 protein, n = 5, P < 0.002) compared to healthy control dogs (95.1 (6.7), n = 22). Alkaline phosphatase, gamma-glutamyl transferase, zinc-resistant alpha-glucosidase, maltase, sucrase and lactase in the ApN deficient dogs exhibited comparable activities to those in the control dogs. Microvillar membranes were analysed by one- and two-dimensional electrophoresis. ApN was represented by a single 145kDa band in all control dogs, identified by immunoblotting and immunoprecipitation. Protein maps from deficient dogs were normal apart from the virtual absence of an ApN spot and there were no apparent abnormalities in the glycosylation of microvillar proteins. The findings suggest that intestinal ApN deficiency in these dogs is a primary lesion involving diminished expression of an otherwise normal enzyme protein.

Longitudinal variation in the activities of mucosal enzymes in the small intestine of suckling rats.

The extent of positional variation in mucosal enzyme activity along the small intestine was investigated in 14-day-old suckling rats. Samples were taken from ten equally spaced sites along the intestine in 11 rat pups and the activities of the enzymes alkaline phosphatase, neutral aminopeptidase, gamma-glutamyl transferase, lactase and sucrase were measured. All the enzymes except sucrase were subject to considerable positional variation. Alkaline phosphatase and aminopeptidase activities were distributed throughout the intestine, with a broad maximum in the distal intestine. Lactase was also broadly distributed but with greatest activity in the mid intestine. gamma-glutamyl transferase exhibited a novel profile, with a very high proportion of the total activity (78%) present in the distal intestine. Sucrase was essentially absent throughout the intestine.

Characterization of microvillar membrane proteins of dog small intestine by two-dimensional electrophoresis.

A method for analysing microgram amounts of microvillar membranes by two-dimensional electrophoresis (protein mapping) is described, and has been used to characterize the microvillar proteins of the small intestine of German shepherd, corgi, and beagle dogs. Detergent-solubilized microvillar membranes were radiolabelled with 14C and separated by isoelectric focussing followed by SDS-PAGE. Proteins were detected fluorographically and glycoproteins by lectin-affinity staining. The microvillar hydrolases alkaline phosphatase and dipeptidyl aminopeptidase IV were identified by active-site labelling and aminopeptidase N by immunoprecipitation. Changes following pancreatic duct diversion were consistent with accumulation of pro-sucrase-isomaltase and diminished expression of the sucrase and isomaltase subunits. Cytoskeletal proteins were concentrated in the core fraction remaining after extraction of microvillar membranes with Triton X-100. There were no consistent differences between dogs of different breed, and the canine protein maps were similar to the human.

Basic fibroblast growth factor increases the multiplication and migration of a serum-free derivative of CACO-2 but does not affect differentiation.

Basic fibroblast growth factor (bFGF) is found in the extracellular matrix and around the endothelial and epithelial cells of some human colon carcinomas. It is believed to play a role in angiogenesis, but in addition, recent data suggest that it can directly stimulate mitogenesis in some colon carcinoma cell lines. To clarify the role of bFGF in human colon carcinoma, we developed a model of Caco-2 which grew in serum-free conditions so that the effect of bFGF on multiplication, migration, and differentiation could be studied in defined conditions. Through morphological and biochemical studies in serum-free conditions, we demonstrated that this subline of Caco-2 differentiated spontaneously on reaching confluence. Using this model, we found that bFGF did not affect differentiation but that multiplication and migration were increased. The implication of these findings is that bFGF, released from the extracellular matrix by invading cells or produced by neovascular endothelial cells, can increase the mitogenic rate and migratory potential of colon carcinoma cells. In addition, the dual role of bFGF in stimulating colon carcinoma cells directly and promoting angiogenesis suggests that anti-bFGF strategies could form the basis of a novel approach to the treatment of colon carcinoma.

Acid-base transport systems in a polarized human intestinal cell monolayer: Caco-2.

Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.

Cytokeratin expression in epithelial cells isolated from the crypt and villus regions of the rodent small intestine.

Many physiological and structural features of epithelium in the small intestine are regulated during their transit from the crypt base to the villus tip. This crypt-villus axis is an important model for the study of the regulation of cell proliferation and differentiation. We have investigated the expression of cytokeratins in purified epithelial cells from the proliferative (crypt) and differentiated (villus) regions of this tissue. Three polypeptides were identified (cytokeratins 8, 18 and 19) as well as a fourth, 46 kDa polypeptide with similar electrophoretic characteristics to the recently identified cytokeratin 20. The distribution of these molecules was found to vary along the crypt-villus axis, with cytokeratin 18 being restricted to the proliferative crypt and cytokeratins 8 and 19 demonstrating more uniform distributions. The 46 kDa component was found to be expressed predominantly within the villus epithelium. Although there is no substantial evidence of a direct role for cytokeratins in the process of epithelial differentiation, these data suggest that differential expression of cytokeratins is associated with changes in intestinal epithelial differentiation.

Effects of urogastrone-epidermal growth factor and age at administration on five enzymes in the small intestine of suckling rats.

Suckling rats were given urogastrone-epidermal growth factor (EGF: 1,000 micrograms/kg body weight) or vehicle by gavage at one of three stages of development: 8 to 10, 11 to 13 or 14 to 16 days of age. Intubation was carried out at 8-hourly intervals over these periods. Fourteen to 16 h after the last intubation the rats were killed; that is, at 11, 14 and 17 days respectively. Samples of proximal and distal small intestine (SI) were taken for enzyme analysis. Five enzymes were assayed; sucrase, lactase, gamma-glutamyl transferase, alkaline phosphatase and neutral amino-peptidase, and their activities expressed per g protein. Treatment with EGF had no effect on body weight or on the length of the small intestine at any age. The nature of the effects on enzyme activities depended on the specific enzyme concerned, the site within the small intestine and the timing of the treatment. Lactase was increased by EGF at both sites only on day 14, whereas gamma-glutamyl transferase was increased in proximal samples at 11 and 14 days, and in distal samples at 17 days. Nor was the outcome always to increase activity. On day 11 alkaline phosphatase was increased in proximal SI, but decreased in distal SI; and so too was aminopeptidase N decreased in distal SI at 11 days. Sucrase showed no response at all. The pattern is complex. Certainly it does not indicate accelerated functional maturation.

Spectrofluorimetric determination of urinary p-aminobenzoic and p-aminosalicylic acids in the BT-PABA/PAS test of pancreatic function.

Spectrofluorimetry was investigated as an alternative to HPLC for determining p-aminobenzoic acid and p-aminosalicylic acid in the N-benzoyl-L-tyrosyl-p-aminobenzoic acid/p-aminosalicylic acid test of pancreatic exocrine function. Urine specimens were hydrolysed for 30 min in 4 M NaOH at 100 degrees C. The fluorescence of p-aminobenzoic acid was measured in dimethyl sulphoxide solution (lambda ex = 300 nm, lambda em = 340 nm) and that of p-aminosalicylic acid in sodium acetate buffer, pH 4.0 (lambda ex = 297 nm, lambda em = 394 nm). The linear range was 0.038-8 mM for p-aminobenzoic acid and 0.051-12 mM for p-aminosalicylic acid, within-batch precision was 2.2% and 5.5%, respectively, and the entire analysis could be completed within 40 min. Although not eliminated, drug interference was greatly reduced in comparison with colorimetry. In 23 consecutive pancreatic function tests there was an excellent correlation between the p-aminobenzoic acid/p-aminosalicylic acid excretion index obtained by fluorimetry and the results from HPLC analysis (y = 0.914x + 0.070, r = 0.987, p less than 0.001). The method is simple, cost-effective and may be particularly valuable in developing countries having a high incidence of chronic pancreatitis.

Investigation of the physical properties of dog intestinal microvillar membrane proteins by polyacrylamide gel electrophoresis: a comparison between normal dogs and dogs with exocrine pancreatic insufficiency.

Procedures have been validated for the investigation of the physical properties of canine microvillar membrane proteins by SDS-polyacrylamide gel electrophoresis. These have been used to examine mucosal samples from eight control dogs and from five dogs with naturally occurring exocrine pancreatic insufficiency (EPI) in order to evaluate the potential role of the pancreas in the normal turnover of microvillar membrane proteins in the dog. Gel scanning showed that the proportion of total membrane protein in bands corresponding to a molecular mass greater than 200 kDa was up to 20-times higher in dogs with EPI than in control dogs. In particular, a band of apparent molecular mass 218 kDa represented between 8 and 28% of membrane protein in all affected dogs, compared with only 0.5 to 1.8% in controls, and is most likely to contain single chains of both pro-maltase-glucoamylase and pro-sucrase-isomaltase. Incubation of microvillar membranes in vitro with either trypsin or canine pancreatic juice resulted in degradation of this high molecular mass band and a corresponding increase in the amount of protein in three bands representing molecular masses of 150, 133 and 106 kDa. In samples from control dogs aminopeptidase N was identified in the 133 kDa band by Western blotting and incubation with monospecific antiserum. These findings suggest that pancreatic enzymes play a major role in the normal post-translational processing of intestinal microvillar membrane proteins in the dog.