Measuring the lamellarity of giant lipid vesicles with differential interference contrast microscopy.

Giant unilamellar vesicles are a widely utilized model membrane system, providing free-standing bilayers unaffected by support-induced artifacts. To measure the lamellarity of such vesicles, fluorescence microscopy is one commonly utilized technique, but it has the inherent disadvantages of requiring lipid staining, thereby affecting the intrinsic physical and chemical properties of the vesicles, and it requires a calibration by statistical analysis of a vesicle ensemble. Herein we present what we believe to be a novel label-free optical method to determine the lamellarity of giant vesicles based on quantitative differential interference contrast (qDIC) microscopy. The method is validated by comparison with fluorescence microscopy on a statistically significant number of vesicles, showing correlated quantization of the lamellarity. Importantly, qDIC requires neither sample-dependent calibration nor sample staining, and thus can measure the lamellarity of any giant vesicle without additional preparation or interference with subsequent investigations. Furthermore, qDIC requires only a microscope equipped with differential interference contrast and a digital camera.

Brillouin-Raman microspectroscopy for the morpho-mechanical imaging of human lamellar bone.

Bone has a sophisticated architecture characterized by a hierarchical organization, starting at the sub-micrometre level. Thus, the analysis of the mechanical and structural properties of bone at this scale is essential to understand the relationship between its physiology, physical properties and chemical composition. Here, we unveil the potential of Brillouin-Raman microspectroscopy (BRaMS), an emerging correlative optical approach that can simultaneously assess bone mechanics and chemistry with micrometric resolution. Correlative hyperspectral imaging, performed on a human diaphyseal ring, reveals a complex microarchitecture that is reflected in extremely rich and informative spectra. An innovative method for mechanical properties analysis is proposed, mapping the intermixing of soft and hard tissue areas and revealing the coexistence of regions involved in remodelling processes, nutrient transportation and structural support. The mineralized regions appear elastically inhomogeneous, resembling the pattern of the osteons' lamellae, while Raman and energy-dispersive X-ray images through scanning electron microscopy show an overall uniform distribution of the mineral content, suggesting that other structural factors are responsible for lamellar micromechanical heterogeneity. These results, besides giving an important insight into cortical bone tissue properties, highlight the potential of BRaMS to access the origin of anisotropic mechanical properties, which are almost ubiquitous in other biological tissues.

Inhibitory effect of luteinising hormone-releasing hormone analogues on human endometrial cancer in vitro.

We studied the effects of luteinising hormone-releasing hormone (LHRH) agonist leuproreline (1 microM for 96 h) and LHRH antagonist cetrorelix on the cell growth of primary cultures from nine human endometrial cancers using the sulphorhodamine colorimetric test. Histological examinations and reverse transcription and polymerase chain reaction amplification (RT-PCR) for LHRH receptors were also performed. The endometrial cancers examined had a medium to high degree of proliferative activity and a low degree of apoptotic power; furthermore, they expressed the LHRH receptor RNA variably, detectable in 71% of cases. The addition of leuproreline or cetrorelix to cell cultures inhibited growth in a statistically significant way compared to untreated control cells; nevertheless, the percentage of cell growth inhibition obtained was very variable. These data suggest that LHRH analogues can exert differential inhibitory effects on the growth of endometrial cancer, which seems to be independent of the expression of specific LHRH receptors.

Separation of coherent and incoherent nonlinearities in a heterodyne pump-probe experiment.

We demonstrate that the transient coherent nonlinearity (coherent artifact) affecting the pump-probe response of semiconductor optical amplifiers can be experimentally separated from the incoherent transient. The technique is based on measuring the mirror component of the coherent artifact which is a background-free four--wave mixing signal at a different frequency with respect to the transmitted probe in a heterodyne detection scheme. Measurements on amplifiers of different length reveal strong deviations from the commonly expected symmetric shape of the coherent artifact in case of long waveguides.

Lectin binding in the human endometrium in early luteal phase following controlled ovarian hyperstimulation.

A lectin histochemical study was performed to investigate the glycoconjugate saccharidic moieties on the endometrial epithelium and stroma in 12 women undergoing controlled ovarian hyperstimulation (COH) for in-vitro fertilisation for embryo transfer (IVF-ET) in early luteal phase. 7 control subjects were also evaluated. For this purpose a battery of seven horseradish peroxidase-conjugated lectins (PNA, SBA, DBA, WGA, ConA, LTA and UEA I) was used. Cytochemical controls were performed for specificity of lectin-sugar reaction. As far as the endometrial glands and stroma are concerned, the obtained data showed no differences in the endometrial lectin binding between the subjects of the control group and the ones undergoing COH, with the exception of PNA reactivity at the level of the apical portion of the glandular cells, which was detected only in COH women. It is noteworthy that, although the endometrial dating using the Noyes's criteria showed marked dissynchronies between the stroma and the glands in COH subjects, a uniformity of lectin binding, revealing the same type and localization of terminal oligosaccharides, was observed in all the examined subjects. The uniformity in distribution of the sugar residues detected in the endometrial specimens following COH might be due to the massive FSH and/or hCG treatment which probably determines an endometrial environment almost equal in all the examined subjects. In all the treated subjects reactivity with sialidase-WGA and ConA, revealing the presence of N-acetyl-D-glucosamine and D-mannose respectively, was detected at the level of the lining epithelium.

Ageing of the human oviduct: lectin histochemistry.

The aim of the present research was to investigate the changes of the sugar residues in the oviduct in the course of ageing in postmenopausal women vs normally menstruating women, by means of lectin histochemistry. Twenty asymptomatic postmenopausal women (48-83 years old) were recruited among patients who underwent a vaginal hysterectomy. Eight normally menstruating women were recruited as controls. Fragments of Fallopian tubes (pars ampullaris) were fixed in 10% formalin and routinely processed. The sections were labelled with HRP-lectins (PNA, SBA, DBA, WGA, Con A, LTA, UEAI). Some sections were pre-treated with neuraminidase prior to staining with HRP-lectins. Among the postmenopausal patients, our histochemical data showed that there was no difference in the localization and distribution of sugar residues of glycoconjugates as detected by various HRP-lectins. Moreover, our results demonstrated that the oviductal epithelium is characterized by apical reactivity in both ciliated and non-ciliated cells. In the course of ageing, the ciliated cells changed their morphology from bathyprismatic to large and rounded shape. ConA lectin reacted intensely with such highly degenerating ciliated cells and could be considered a marker of these cells. The degenerating ciliated cells are also characterized by the absence of sialic acid. In comparison with the sugar residues present in the control group, the oviductal epithelium of postmenopausal women is characterized by the loss of reactivity with DBA, WGA and ConA. Moreover, PNA reactive material was present at the free border of the ciliated and non-ciliated cells. The latter findings were statistically confirmed and could be considered strictly related to the ageing process.

Decidual progesterone and estrogen receptors in the first trimester of pregnancy.

Receptor content of human decidua in early pregnancy (weeks 6-12) was investigated. Fifty-three tissue samples were obtained from voluntary patients undergoing abortion and whose gestational age range from 6 to 12 weeks. Blood samples were drawn at the time of operation in order to measure circulating estradiol (E) and progesterone (P) concentrations. Tissue samples underwent first histological confirmation and then were analyzed for receptor content by immunohistochemistry (IH) and by the conventional ligand binding technique (LBA). Estrogen receptors (ER) appeared to be always undetectable by IH (53 samples). LBA measured a significant amount of ER (> 10 fmol/mg) in two samples, borderline values (3-10 fmol/mg) in 6 and no binding in the other three. No relation was apparent between PR levels and either gestational age or blood P concentration. ER were possibly downregulated by the high E levels, and their synthesis inhibited by the high P levels.

Maternal serum CA 125 levels in first trimester abortion.

OBJECTIVE: To assess the source of maternal serum CA 125 during the first trimester of pregnancy. STUDY DESIGN: CA 125 was measured in stored samples from nonviable pregnancies of 8-13 weeks gestation. The study group comprised 19 women with vaginal bleeding and 13 non-bleeders. Only patients in whom chromosome analysis of the products of conception demonstrated a normal caryotype were included. CA 125 levels were expressed in multiples of the median (MoM) for normal pregnancies of the same gestational age. RESULTS: Median MoM values of CA 125 were significantly higher in women with vaginal bleeding (1.81 MoM) as compared both to non-bleeders (0.82 MoM; p < 0.01-Mann-Whitney U-test) and to the normal pregnancies (1.01 MoM; p < 0.05). No significant difference was found between non-bleeding women and controls. CONCLUSIONS: The present study indicates that in non-viable pregnancies with euploid fetuses an increase in maternal serum CA 125 levels was found only in presence of decidual disruption associated to vaginal bleeding. These findings are compatible with a prevalent decidual source of this antigen.

I. Aging of the human endometrium: a basic morphological and immunohistochemical study.

OBJECTIVE: To evaluate if human endometrium presents morphological variations suggestive of an age-related decline in endometrial receptivity. STUDY DESIGN: Peri-implantation endometrium of younger (<30 years of age: n = 13) and older (>40 years of age: n = 17) normally menstruating women was studied. Endometrial specimens were routinely fixed in buffered formalin and embedded in paraffin. Sections (5 mu m) were stained with hematoxylin-eosin, periodic acid-Schiff (PAS) and Trichrome conforming to Masson according to conventional histologic examination. Several consecutive sections were used for the following immunohistochemical study: vascular localization (CD34), cellular proliferation index (PCNA), progesterone and estrogen receptors. RESULTS: Using both the traditional morphological evaluation and monoclonal antibodies, no significant differences were found between the endometria of women <30 years of age and those of women >40. CONCLUSIONS: Our results suggest that human endometrium does not age, at least while cyclic hormonal stimulation and menstruation are present.

Aging of the human endometrium: peri-implantation phase endometrium does not show any age-dependent variation in lectin binding.

OBJECTIVE: To evaluate if the peri-implantation endometrium shows age variations in lectin patterns, which suggest possible age variations in embryo-maternal recognition. STUDY DESIGN: Peri-implantation endometria of younger ( < 30 years of age: n = 13) and older ( > 40 years of age: n = 17) normally menstruating women was studied. Endometrial specimens were routinely fixed in buffered formaline and embedded in paraffin. Sections (5 microns) were studied using seven lectins: DBA (Dolicus biflorus, binding specificity alpha-D-GalNAc), PNA (Arachis hypogea, binding specificity D-Gal (beta 1 --> 3)-D-GalNAc), SBA (Glycine max binding specificity alpha/beta-D-GalNAc > D-Gal), WGA (Triticum vulgare binding specificity (alpha-D-GlcNAc)n and sialic acid), ConA (Canavalia ensiformis binding specificity alpha-D-Man > alpha-D-Glc), LTA (Lotus tetragonolobus binding specificity alpha-L-fucose) and UEA 1 (Ulex europaeus binding specificity alpha-L-fucose). RESULTS: No significant differences were found in the glycoconjugates sugar residue content and distribution between the endometria of women < 30 years of age and those of women > 40. CONCLUSIONS: Our results suggest that human endometrium does not age, at least while cyclic hormonal stimulation and menstruation are present.

Hormonal patterns, steroid receptors and morphological pictures of endometrium in hyperstimulated IVF cycles.

OBJECTIVE: The purpose of this contribution is to investigate the pathophysiology of the abnormal endometrial development in hyperstimulated IVF cycles. STUDY DESIGN: In 12 IVF-patients who did not have embryo transfer because of failure of oocyte fertilization, serum values of 17 beta-estradiol, progesterone, FSH, LH, total and free testosterone, and androstenedione were measured on the pick-up day and were evaluated with respect to the values normally expressed in the day of ovulation; in the endometrial specimens collected 2 days later, at the time of embryo replacement, estrogen and progesterone receptors were immunohistochemically determined and dating by the Noyes method was performed. RESULTS: 17 beta-Estradiol values are constantly higher, and progesterone levels are, only in four cases, higher than expected for the day of ovulation in a natural cycle. These hormonal patterns can only partially explain the pattern of steroid receptors: progesterone receptors are expressed sparsely both in glands and stroma, while estrogen receptors are abundant in the glands and absent in the stroma. In 11 of 12 patients an abnormal endometrial development with stromal advancement was observed: this morphological picture of the endometrium could partially be explained only in the four cases presenting high progesterone levels by serum values and endometrial receptor content of estrogen and progesterone. CONCLUSIONS: The abnormal endometrial development in hyperstimulated IVF cycles could only in part be explained by estrogen and progesterone, and other factors have to be considered.

[Cardiotocographic monitoring of fetal health. Comparative evaluation of traditional and computerized methods]. / Monitoraggio cardiotocografico del benessere fetale. Valutazione comparativa fra la metodica tradizionale e quella computerizzata.

One hundred physiological pregnant women and 76 pathological pregnant women suffering from gestational diabetes and pregnancy-induced hypertension underwent a cardiotocographic examination during the course of routine diagnostic tests. The interpretation of cardiotocographic printouts was carried out using traditional as well as computerised methods. The outcome of these tests was then related to neonatal outcome and other parameters which contributed to defining the prognosis of pregnancy. Computerised analysis was found to provide a more reliable diagnosis in comparison to traditional methods in identifying those pregnancies with a pathological neonatal outcome. In particular, in the group of physiological pregnancies, computerised interpretation proved more reliable in 87.5% of cases in which neonatal outcome was pathological; on the contrary, traditional interpretations only revealed 37.5% of the same cases. In pathological pregnancies, automatic interpretation was also found to be more reliable in predicting the non-pathological outcomes, whereas traditional methods provided a high incidence of uncertain answers.