Concentrations of homocysteine in follicular fluid and embryo quality and oocyte maturity in infertile women: a prospective cohort.

Homocysteine is one of the components of follicular fluid (FF), so that any disruptions in its concentration may affect oocyte development. The aim of this study was to determine the relationship between FF homocysteine concentration and embryo quality, oocyte maturity, and pregnancy rate. Oocytes and embryos of 44 infertile women were categorised into different groups based on their maturity and quality, respectively. FF homocysteine levels, oocyte maturity, embryo quality, and pregnancy status were measured. A significant association was observed between the levels of FF homocysteine ​and oocyte maturation rate (p = .00). The concentration of FF homocysteine was higher than 9.8 µm/L in women with oocyte maturation < 80%. Most of the good quality embryos belonged to homocysteine levels < 9.8 µm/L. Decreased FF homocysteine concentrations can significantly improve the oocyte maturation rate and embryo quality. Aging may be an indirect factor contributing to decreased embryo quality and oocyte maturation through increasing FF homocysteine levels.IMPACT STATEMENTWhat is already known on this subject? It has been demonstrated that homocysteine is one of the components of follicular fluid (FF), but no information is available about the link between its concentration in FF and oocyte development.What do the results of this study add? The data indicated that decreased FF homocysteine concentrations at a younger age may remarkably improve the oocyte maturity and embryo quality of infertile patients undergoing assisted reproductive treatment (ART).What are the implications of these findings for clinical practice and/or further research? Based on the findings and considering the ease of measuring serum homocysteine and its direct correlation with FF homocysteine, homocysteine level measurement is recommended in patients who are candidates for infertility treatment in order to estimate oocyte maturation rate, embryo quality, and ART outcomes. Future studies are suggested to investigate patients with PCOS, endometriosis, and male factor infertility.

Predischarge monitoring of preterm infants.

The objectives of this study were: 1) to perform documented event-monitoring (DEM) for apnea (A, > or = 20 s) and bradycardia (B, < 80 beats per min for > or = 5 s) in premature infants prior to discharge, and 2) to examine the accuracy of nursing documentation (ND) of A and B. Forty-four stable preterm infants, with mean weights and gestational ages at birth (+/- SD) of 1,543 (+/- 365) g, and 30 (+/- 2) weeks, respectively, were studied using DEM for 9 (+/- 2) days prior to discharge. Differences in DEM and ND were analyzed by the z-test for proportions. There were 561 true events recorded by DEM: 56 were As and 505 were Bs. ND revealed 296 events, 190 As and 106 Bs. Of the 56 true As on DEM, only 21 (38%) were correctly reported by ND (P < 0.001, 95% confidence interval (CI) 0.44-0.81). Of the 505 true Bs on DEM, 153 (30%) were correctly reported by ND (P < 0.001, CI 0.63-0.76). When ND was compared with DEM, 174 (59%) of NDs were true events. Of the 106 As on ND, only 21 (20%) were true As on DEM (P < 0.001, CI 0.58-1). Of the 190 Bs on ND, 153 (80%) were true Bs on DEM (P < 0.001, CI 0.13-0.26). ND did not detect 6 of the 33 infants who had significant events on DEM, while 4 of the 11 who had events reported on ND did not have any on DEM. Thus, 10 infants were misclassified by ND (P < 0.01, CI 0.1-0.36). These results indicate that, compared to DEM, ND not only identified significantly fewer true As and Bs, but also misclassified a significant number of infants. We conclude that DEM performed prior to discharge for preterm infants at risk for apnea and bradycardia provides more objective and accurate information than ND.

Heparin-like compounds prepared by chemical modification of capsular polysaccharide from E. coli K5.

O-Sulfation of sulfaminoheparosan SAH, a glycosaminoglucuronan with the structure-->4)-beta-D-GlcA(1-->4)-beta-D-GlcNSO3(-)-(1-->, obtained by N-deacetylation and N-sulfation of the capsular polysaccharide from E. coli K5, was investigated in order to characterize the sulfation pattern eliciting heparin-like activities. SAH was reacted (as the tributylammonium salt in N,N-dimethylformamide) with pyridine-sulfur trioxide under systematically different experimental conditions. The structure of O-sulfated products (SAHS), as determined by mono- and two-dimensional 1H and 13C NMR, varied with variation of reaction parameters. Sulfation of SAH preferentially occurred at O-6 of the GlcNSO3- residues. Further sulfation occurred either at O-3 or at O-2 of the GlcA residues, depending on the experimental conditions. Products with significantly high affinity for antithrombin and antifactor Xa activity were obtained under well-defined conditions. These products contained the trisulfated aminosugar GlcNSO3-3,6SO3-, which is a marker component of the pentasaccharide sequence through which heparin binds to antithrombin.

Biosynthesis of heparin/heparan sulfate. The D-glucosaminyl 3-O-sulfotransferase reaction: target and inhibitor saccharides.

O-Sulfation at C-3 of N-sulfated GlcN units concludes polymer modification and the formation of antithrombin binding regions in the biosynthesis of heparin/heparan sulfate. The resulting GlcNSO3(3-OSO3) units are largely restricted to heparin chains with high affinity for antithrombin (HA heparin). Low affinity (LA) heparin fails to serve as a substrate in the 3-O-sulfotransferase reaction yet contains potential 3-O-sulfate acceptor sites (Kusche, M., Torri, G., Casu, B., and Lindahl, U. (1990) J. Biol. Chem. 265, 7292-7300), as verified in the present study using a novel sequencing procedure. O-Desulfated, re-N-sulfated LA heparin, as well as an octasaccharide fraction isolated after heparinase I digestion of LA heparin, both yielded labeled HA components following incubation with solubilized mouse mastocytoma microsomal enzymes and [35S]adenosine 3'-phosphate 5'phosphosulfate (PAPS), suggesting that the 3-O-sulfo-transferase may be inhibited by sulfated saccharide sequences outside the 3-O-sulfate acceptor region. Indeed, the addition of LA heparin precluded enzymatic 3-O-sulfation of a synthetic pentasaccharide substrate. The Km for the pentasaccharide was determined to approximately be 6 microM. Incubations of mixed pentasaccharide substrate and saccharide inhibitors revealed Ki values for intact LA heparin and for a heparin octasaccharide fraction of approximately 1.3 and approximately 0.7 microM, respectively. Inhibition experiments with selectively desulfated heparin indicated that both IdoA 2-O-sulfate and GlcN 6-O-sulfate groups contributed to the inhibition of the 3-O-sulfotransferase. By contrast, chondroitin sulfate or dermatan sulfate showed no significant inhibitory activity. It is proposed that the regulation of GlcN 3-O-sulfation during biosynthesis of heparin/heparan sulfate depends on the topological organization of the membrane-bound enzyme machinery in the intact cell.

Masking and unmasking of the sialic acid-binding lectin activity of CD22 (Siglec-2) on B lymphocytes.

CD22 is a B cell-restricted glycoprotein involved in signal transduction and modulation of cellular activation. It is also an I-type lectin (now designated Siglec-2), whose extracellular domain can specifically recognize alpha2-6-linked sialic acid (Sia) residues. This activity is postulated to mediate intercellular adhesion and/or to act as a coreceptor in antigen-induced B cell activation. However, studies with recombinant CD22 indicate that the lectin function can be inactivated by expression of alpha2-6-linked Sia residues on the same cell surface. To explore whether this masking phenomenon affects native CD22 on B cells, we first developed a probe to detect the lectin activity of recombinant CD22 expressed on Chinese hamster ovary cells (which have no endogenous alpha2-6-linked Sia residues). This probe is inactive against CD22-positive B lymphoma cells and Epstein-Barr virus-transformed lymphoblasts which express high levels of alpha2-6-linked Sia residues. Enzymatic desialylation unmasks the CD22 lectin activity, indicating that endogenous Sia residues block the CD22 lectin-binding site. Truncation of the side chains of cell surface Sia residues by mild periodate oxidation (known to abrogate Sia recognition by CD22) also had this unmasking effect, indicating that the effects of desialylation are not due to a loss of negative charge. Normal resting B cells from human peripheral blood gave similar findings. However, the lectin is partially unmasked during in vitro activation of these cells. Thus, the lectin activity of CD22 is restricted by endogenous sialylation in resting B cells and may be transiently unmasked during in vivo activation, perhaps to modulate intercellular or intracellular interactions at this critical stage in the humoral response.

Cryptic sialic acid binding lectins on human blood leukocytes can be unmasked by sialidase treatment or cellular activation.

We recently reported that the sialic acid-specific binding sites of CD22 molecules on B cells are masked by endogenous ligands, and can be unmasked by sialidase treatment or cellular activation. Here, we show that many other human blood leukocyte types have endogenous sialic acid binding sites that can be unmasked by sialidase treatment. Truncation of sialic acid side chains on the soluble probes used for detection abolishes all binding, indicating the specificity of the interaction for the details of sialic acid structure. There is limited overlap between alpha2-6- and alpha2-3-sialic acid-specific binding sites, which are unmasked on monocytes, natural killer cells, a minority of mature T cells, neutrophils, and some cultured human leukemic cell lines. Activation with phorbol ester and calcium ionophore causes spontaneous exposure of some of the binding sites, occurring over a period of minutes on neutrophils and several hours on monocytes and U937 leukemia cells. Activation is accompanied by some evidence for desialylation of cell surface molecules. Thus, many human blood cells have specific binding sites for sialic acids, masked by endogenous sialylated ligands. Cellular activation can unmask these sites, possibly by the action of an endogenous sialidase. The nearly universal masking of such sites in unactivated blood cells could explain why many of these sialic acid-binding lectins have not been previously discovered. Similar considerations may apply to sialic acid binding lectins of other cell types and tissues.

Limb gangrene secondary to thromboembolic disease of the newborn.

Thromboembolic disease of the newborn is a well-known entity to neonatologists. When severe arterial compromise threatens limb survival, the orthopaedic surgeon may be called on to assist in the management of this disorder. A case of spontaneous postnatal ischemic gangrene of an upper extremity that resulted in amputation in a premature infant is presented; management options are discussed. Newborns are prone to thromboembolism due to a fibrinolytic capability that is significantly different from that of adults.

Structural and functional properties of heparin analogues obtained by chemical sulphation of Escherichia coli K5 capsular polysaccharide.

Capsular polysaccharide from Escherichia coli K5, with the basic structure (GlcA beta 1-4GlcNAc alpha 1-4)n, was chemically modified through N-deacetylation, N-sulphation and O-sulphation [Casu, Grazioli, Razi, Guerrini, Naggi, Torri, Oreste, Tursi, Zoppetti and Lindahl (1994) Carbohydr. Res. 263, 271-284]. Depending on the reaction conditions, the products showed different proportions of components with high affinity for antithrombin (AT). A high-affinity subfraction, M(r) approx. 36,000, was shown by near-UV CD, UV-absorption difference spectroscopy and fluorescence to cause conformational changes in the AT molecule very similar to those induced by high-affinity heparin. Fluorescence titrations demonstrated about two AT-binding sites per polysaccharide chain, each with a Kd of approx. 200 nM. The anti-(Factor Xa) activity was 170 units/mg, similar to that of the IIId international heparin standard and markedly higher than activities of previously described heparin analogues. Another preparation, M(r) approx. 13,000, of higher overall O-sulphate content, exhibited a single binding site per chain, with Kd approx. 1 microM, and an anti-(Factor Xa) activity of 70 units/mg. Compositional analysis of polysaccharide fractions revealed a correlation between the contents of -GlcA-GlcNSO3(3,6-di-OSO3)- disaccharide units and affinity for AT; the 3-O-sulphated GlcN unit has previously been identified as a marker component of the AT-binding pentasaccharide sequence in heparin. The abundance of the implicated disaccharide unit approximately equalled that of AT-binding sites in the 36,000-M(r) polysaccharide fraction, and approached one per high-affinity oligosaccharide (predominantly 10-12 monosaccharide units) isolated after partial depolymerization of AT-binding polysaccharide. These findings suggest that the modified bacterial polysaccharide interacts with AT and promotes its anticoagulant action in a manner similar to that of heparin.

Identification of O-sulphate substituents on D-glucuronic acid units in heparin-related glycosaminoglycans using novel synthetic disaccharide standards.

The two disaccharides, methyl 4-O-(2-O-sulpho-beta-D-glucopyranosyl-uronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside and methyl 4-O-(3-O-sulpho-beta-D-glucopyranosyluronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside, were prepared by de novo synthesis, and converted to the corresponding 2,5-anhydro-D-[1-3H]mannitol derivatives by deamination with nitrous acid followed by reduction with NaB3H4. The resultant labelled products were used as standards in the identification, by anion-exchange high-performance liquid chromatography (HPLC), of disaccharides generated by HNO2/NaB3H4 treatment of heparan sulphate isolated from human brain. The two standards, containing 2-O- and 3-O-sulphated glucuronic acid, respectively, were clearly separated by the HPLC procedure. Comparison with the deamination products derived from heparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sulphated standard. The corresponding component isolated from other heparan sulphate preparations, or from heparin, also eluted at the same position. No disaccharide derived from heparin or heparan sulphate appeared at the elution position of the 3-O-sulphated standard. It is concluded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulphation at C3. By contrast, analysis of mono-O-sulphated disaccharides derived from a chemically sulphated, bacterial capsular polysaccharide (generated by Escherichia coli K5) clearly demonstrated the occurrence of O-sulphate groups at C-3 of D-glucuronic acid units.