Ovarian stimulation for in-vitro fertilization combining administration of gonadotrophins and blockade of the pituitary with D-Trp6-LHRH microcapsules: pilot studies with two protocols

In women undergoing in-vitro fertilization and embryo transfer(TVF-ET), a total of 408IVF cycles were stimulated using humanmenopausal gonadotrophin (HMG) or pure follicle stimulatinghormone (FSH) plus HMG in combination with a single injectionof D-Trp⁶-LHRH microcapsules in order to enhance the ovarianresponse to gonadotrophins and to avoid spontaneous LH surges.Sixty-seven pregnancies were achieved. Two protocols were employed.In protocol 1 (‘blocking protocol’, n = 268), thepituitary was first inhibited with a full dose (3.75 mg) ofD-Trp⁶-LHRH in microcapsules and ovarian stimulation was startedafter the hypogonadotrophic hypogonadal state was ascertained(Ej >50 pg/ml). In protocol 2 (‘flareup protocol’,n = 140), the treatment with D–Trp⁶LHRH microcapsules(half-dose = 1.80 mg) and the ovarian stimulation with gonadotrophinswere started at the same time. Higher doses of gonadotrophinswere needed (39.5 11.2 ampoules FSH and/or HMG) in protocol1, in which the pituitary was blocked prior to and during thestimulation, than in protocol 2 (209 ampoules) where the exogenousgonadotrophin stimulation appeared to be augmented by the initialagonistic effect of the injection of D-Trp⁶LHRH microcapsules.In patients with purely tubal infertility, under 38 years oldand no male factor, the results obtained with protocols 1 and2 were similar in terms of pregnancy rate per cycle or per embryotransfer: 22.6 versus 20.5% and 28.3 versus 27.4%, respectively.However, considering the cost benefit, ‘flare-up’protocols appeared to be a better choice and could be recommended.     

Light scattering from normal and dysplastic cervical cells at different epithelial depths: finite-difference time-domain modeling with a perfectly matched layer boundary condition

The finite-difference time-domain (FDTD) method provides a flexible approach to studying the scattering that arises from arbitrarily inhomogeneous structures. We implemented a three-dimensional FDTD program code to model light scattering from biological cells. The perfectly matched layer (PML) boundary condition has been used to terminate the FDTD computational grid. We investigated differences in angle-dependent scattering properties of normal and dysplastic cervical cells. Specifically, the scattering patterns and phase functions have been computed for normal and dysplastic cervical cells at three different epithelial depths, namely, basal/parabasal, intermediate, and superficial. Construction of cervical cells within the FDTD computational grid is based on morphological and chromatin texture features obtained from quantitative histopathology. The results show that angle-dependent scattering characteristics are different not only for normal and dysplastic cells but also for cells at different epithelial depths. The calculated scattering cross-sections are significantly greater for dysplastic cells. The scattering cross-sections of cells at different depths indicate that scattering decreases in going from the superficial layer to the intermediate layer, but then increases in the basal/parabasal layer. This trend for epithelial cell scattering has also been observed in confocal images of ex vivo cervical tissue.     

Reducing the time of sperm-oocyte interaction in human in-vitro fertilization improves the implantation rate

Human oocyte development was evaluated after a reduced timeexposure to spermatozoa in vitro. A total of 119 patients wereassigned to two study groups in a randomized prospective studyin which each patient‘s oocytes were exposed to spermatozoafor either 1 h (group 1 – 58 patients) or the standard16 h incubation period (group 2 – 61 patients). The fertilizationrate obtained in group 1 was higher than in group 2 (285/393,73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyteinteraction occurs within 1 h. This was confirmed in a studyin vitro using fluorescently labelled spermatozoa and normaloocyte-cumulus complexes. Spermatozoa enter the cumulus complexwithin 15 min, traverse the cumulus layer within 3 h, and firstappear in the oocyte cortex at 4 h post-insemination. The incidenceof polyspermy was higher in oocytes exposed to spermatozoa for16 h (3%) than for 1 h (1%). There was no difference in thecleavage rate or morphological characteristics of embryos fromboth study groups. However, when evaluating the timing of embryodevelopment, group 1 generated a significantly higher percentageof four to five cell embryos when compared to group 2 (55 versus39%; P < 0.001), documented at 40 h post-insemination. Theimplantation and pregnancy rates for group 1 were 11 and 28%,while the corresponding rates for group 2 were 8 and 15%. Thissuggests that a reduced exposure of oocyte to spermatozoa favoursembryo viability, possibly due to a decrease in potential damagefrom sperm metabolic waste products.     

Analytical model to describe fluorescence spectra of normal and preneoplastic epithelial tissue: comparison with Monte Carlo simulations and clinical measurements

Fluorescence spectroscopy has shown promise for the detection of precancerous changes in vivo. The epithelial and stromal layers of tissue have very different optical properties; the albedo is relatively low in the epithelium and approaches one in the stroma. As precancer develops, the optical properties of the epithelium and stroma are altered in markedly different ways: epithelial scattering and fluorescence increase, and stromal scattering and fluorescence decrease. We present an analytical model of the fluorescence spectrum of a two-layer medium such as epithelial tissue. Our hypothesis is that accounting for the two different tissue layers will provide increased diagnostic information when used to analyze tissue fluorescence spectra measured in vivo. The Beer-Lambert law is used to describe light propagation in the epithelial layer, while light propagation in the highly scattering stromal layer is described with diffusion theory. Predictions of the analytical model are compared to results from Monte Carlo simulations of light propagation under a range of optical properties reported for normal and precancerous epithelial tissue. In all cases, the mean square error between the Monte Carlo simulations and the analytical model are within 15%. Finally, model predictions are compared to fluorescence spectra of normal and precancerous cervical tissue measured in vivo; the lineshape of fluorescence agrees well in both cases, and the decrease in fluorescence intensity from normal to precancerous tissue is correctly predicted to within 5%. Future work will explore the use of this model to extract information about changes in epithelial and stromal optical properties from clinical measurements and the diagnostic value of these parameters.     

Proteomic and postproteomic characterization of keratan sulfate-glycanated isoforms of thyroglobulin and transferrin uniquely elaborated by papillary thyroid carcinomas

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KS-bearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with alpha (2-3)-linked N-acetylneuraminic acid capping residues; and 4) a novel unidentified oligosaccharide moiety at the nonreducing terminus. Comparative analysis of the relative distribution of transferrin in benign versus PTC tissues highlighted a marked malignancy-associated abundance of the molecule, with a >75% frequency in expression in PTC. These findings demonstrate that PTC cells synthesize unique post-translationally modified thyroglobulin and transferrin variants in situ that may be directly exploitable for diagnosis, through histological and noninvasive cytological procedures; for devising novel strategies for antibody-guided imaging of this tumor in vivo; and for postsurgery follow-up of PTC patients.     

Xylitol formation by Candida guilliermondii in media containing different nitrogen sources

The xylose conversion into xylitol by Candida guilliermondii was evaluated in semi-synthetic media supplemented with different nitrogen sources in a ratio C/N equal 25.6. It was noticed that the xylitol yield was around 80% and also that the type of nitrogen source did not influence this bioconversion.     

Improved survival of ischemic cutaneous and musculocutaneous flaps after vascular endothelial growth factor gene transfer using adeno-associated virus vectors

A major challenge in reconstructive surgery is flap ischemia, which might benefit from induction of therapeutic angiogenesis. Here we demonstrate the effect of an adeno-associated virus (AAV) vector delivering vascular endothelial growth factor (VEGF)165 in two widely recognized in vivo flap models. For the epigastric flap model, animals were injected subcutaneously with 1.5 x 10(11) particles of AAV-VEGF at day 0, 7, or 14 before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF was injected intramuscularly. The delivery of AAV-VEGF significantly improved flap survival in both models, reducing necrosis in all treatment groups compared to controls. The most notable results were obtained by administering the vector 14 days before flap dissection. In the transverse rectus abdominis musculocutaneous flap model, AAV-VEGF reduced the necrotic area by >50% at 1 week after surgery, with a highly significant improvement in the healing process throughout the following 2 weeks. The therapeutic effect of AAV-VEGF on flap survival was confirmed by histological evidence of neoangiogenesis in the formation of large numbers of CD31-positive capillaries and alpha-smooth muscle actin-positive arteriolae, particularly evident at the border between viable and necrotic tissue. These results underscore the efficacy of VEGF-induced neovascularization for the prevention of tissue ischemia and the improvement of flap survival in reconstructive surgery.