OBJECTIVE: Endometriosis is a debilitating disease marked by recurrent gynecological proliferations. The present study aimed at performing differential proteomic analysis of matched eutopic and ectopic endometrium from women with ovarian endometriosis.
METHODS: Proteomes were resolved using nano LC-MS and further identified and quantified using ProteinLynx Global SERVER (PLGS) software. Selected proteins were further chosen for validation by real time-polymerase chain reaction (RT-PCR).
RESULTS: The protein profiles uncovered several differentially expressed proteins in the diseased sample (ectopic endometrium) as compared to the reference sample (eutopic endometrium). The study involved an advanced proteomic approach, nano LC-MS, and validates for the first time the upregulation of Mimecan and Lumican proteins in endometriosis.
CONCLUSIONS: These proteins may hence prove as potentially useful tools in the search for diagnostic markers for early detection of the disease.

OBJECTIVE: The aim of the study is to report successful outcome (live births) after sperm sorting with annexin V-MACS on cryopreserved spermatozoa with high level of sperm DNA fragmentation from a cancer patient survivor.
METHODS: Cryopreserved spermatozoa were sorted with annexin V-MACS prior to ICSI. Sperm DNA fragmentation was evaluated by SCSA(®) and TUNEL.
RESULTS: The couple had two previous IVF/ICSI cycles failures using sperm cryopreserved before cancer treatment. On third ICSI cycle attempt results were as follow: pre-annexin V-MACS sperm quality: 10 × 10(6)/ml, 3.3 % progressive motility, 1 % normal forms, TUNEL: 72.5 % positive cells, SCSA(®): 76.6 % DFI. Post-annexin V-MACS sperm quality: 2.8 × 10(6)/ml, 10 % progressive motility, TUNEL: 58.8 % positive cells. Eight metaphase II oocytes were collected, 4 fertilized, 2 embryos were transferred on day 3 and healthy twins were born (1 boy, 1 girl).
CONCLUSIONS: Annexin V-MACS technique could be a potential tool to improve sperm quality on cryopreserved spermatozoa of cancer patient and improve ICSI outcome.

OBJECTIVE: To evaluate the presence of merocyanine 540 (M540) bodies and their impact on the measurement of apoptotic biomarkers in human spermatozoa.
METHODS: Case-control, prospective study.
METHODS: Academic centers.
METHODS: Fertile and subfertile subjects.
METHODS: Semen samples from subfertile and fertile men, 11 per group, were analyzed for basic semen parameters and early (annexin-V binding) and late (terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]) sperm apoptotic biomarkers by flow cytometry. Samples were also stained with M540 to assess the presence of M540 apoptotic bodies.
METHODS: Presence of M540 apoptotic bodies.
RESULTS: Groups differed significantly in the expression of early and late apoptosis biomarkers. The percentage of M540 bodies between groups was not different. The exclusion of M540 bodies from TUNEL results did not have a significant impact on measurement in either fertile or subfertile groups.
CONCLUSIONS: This study confirmed the occurrence of M540 bodies in semen and that male factor infertility is associated with an increased expression of apoptosis biomarkers. Moreover, we demonstrated that the presence of M540 bodies did not affect the quantification of apoptotic biomarkers in either group.

We report a novel strategy for reversible immobilization of diffusive membrane-associated proteins in a native orientation using a liquid-gel bilayer phase transition, and its application to the single molecule study of Cy3-labeled Annexin V (A5) monomers on supported lipid bilayers containing phosphatidylserine (PS) in a Ca(2+)-rich environment. Total internal reflection fluorescence single molecule trajectory analysis revealed that, at low membrane occupancy, A5 monomers diffuse randomly on liquid phase bilayers and occasionally collide with other A5 monomers to form short-lived pseudodimers. During the liquid-to-gel bilayer phase transition, diffusive A5 monomers become immobilized mostly as isolated monomers, with some percentage of dimers and trimers. The EDTA-induced unbinding of immobilized Cy3-A5 spots indicates that Ca(2+)-bridges between A5 and PS lipids are preserved in the immobilized A5 monomers, confirming their native orientation on gel phase bilayers. Furthermore, the persistence of Ca(2+)-bridges during the liquid-to-gel phase transition, despite negligible A5 binding affinity to gel phase bilayers, strongly suggests the formation of tightly bound A5(Ca2+)m(PS)n complexes that diffuse and become immobilized as single units during the bilayer phase transition.

Recombinant factor VIIa (rFVIIa) has been shown to be efficient for the treatment of haemorrhages in patients with Glanzmann\'s thrombasthenia presenting anti-glycoprotein IIb-IIIa antibodies, but the mechanism of action is not well established and there is no routine laboratory test for the monitoring of rFVIIa. In this study, thrombin generation (TG) test was used to assess the efficacy of rFVIIa ex vivo in a Glanzmann patient with inhibitor, who had a surgery for cholesteatoma. The day before surgery, TG capacity in platelet rich plasma was significantly diminished (Endogenous thrombin potential = 637nM x min) in comparison with the normal control group (1338+/-353 nM x min). Thirty minutes after the first infusion of 90 microg/kg of rFVIIa, TG was increased by 59% (1010 nM x min). rFVIIa was administered as intravenous bolus injection of 90 microg/kg q x 2h during the first 24h, than 66microg/kg q x 2h during 24h and 53 microg/kg q x 2h on the post-operative day 3. Residual TG capacity measured before rFVIIa administration mostly remained above 1000nM x min and the coagulation capacity was not significantly modified after a new injection of rFVIIa. The fibrin network was studied with 3D confocal microscopy using clots obtained with TG test. After rFVIIa infusion, the fibrin network was tighter in comparison with the sample before rFVIIa injection. These results provide further ex vivo evidence on haemostatic efficacy of rFVIIa in Glanzmann\'s patients.

Whereas antiphospholipid antibodies (aPL) are associated with thrombotic events and recurrent spontaneous abortion (RSA), the contribution of anti-beta2 glycoprotein 1 (beta2GP1) and anti-annexin V antibodies as risk factors for RSA remain poorly understood. We investigated anti-beta2-GPI and anti-annexin V IgM and IgG antibodies as potential risk factors for RSA in 200 women with more than three consecutive idiopathic RSA, and 200 age-matched, healthy, parous women. Pearson\'s chi squared test analysis showed that while anti-beta2-GPI IgG (P = 0.416) and IgM (P = 0.72) were comparable between patients and controls, elevated anti-annexin V IgG (P = 0.006), but not IgM (P = 0.084), was more pronounced in patients. Higher frequencies of elevated IgG-only (P = 0.005), but not IgM-only (P = 1.000; OR = 6.66), anti-annexin V antibodies were noted among patients. Multinomial regression analysis showed that body-mass index (overweight and obesity; P = 0.008), education status (P < 0.001) and anti-beta2-GPI IgM (P = 0.033), but not IgG (P = 0.723), were associated with early abortion, while anti-beta2-GPI IgG (P = 0.030) and anti-annexin V IgG (P = 0.004) were associated with late RSA. For combined early-late RSA, the only variable selected was education status (P < 0.001), and neither anti-annexin V nor anti-beta2-GPI IgM and IgG was associated with early-late RSA. Accordingly, anti-annexin V and anti-beta2-GPI should be regarded as independent risk markers of RSA.

暂无摘要。

The effects of Candida albicans on sperm parameters and the outcome of infertility treatment are unclear. This report describes a lack of fertilization after assisted reproductive techniques and increased sperm DNA fragmentation in an infertile patient with male accessory gland infection due to Candida albicans. He had normal sperm parameters and, therefore, underwent conventional IVF for a female factor of infertility. No spermatozoa or only one spermatozoon per oocyte were found attached to the zona pellucida of the six mature oocytes retrieved. A new semen sample was then requested from the patient to perform intracytoplasmic sperm injection (ICSI) on the same oocytes, but again no fertilization resulted. Candida albicans was detected in the medium where spermatozoa were co-incubated with oocytes and subsequently in the urethral swabs. It did not have any detrimental effect on sperm parameters soon after ejaculation or following separation of motile spermatozoa by swim-up technique. Fertilization failure after assisted reproduction treatment was associated with an increased percentage of motile spermatozoa having chromatin packaging abnormalities, externalization of phosphatidylserine and DNA fragmentation. In conclusion, Candida albicans did not affect sperm parameters, but increased sperm chromatin packaging damage and apoptosis that might have caused fertilization failure after assisted reproduction treatment in this couple.

暂无摘要。

We have found using imaging techniques that stimulating Jurkat human leukaemic T-cells with ionomycin in the presence of FM1-43, a dye used to monitor exocytosis and endocytosis, causes large (6--10-fold) increases in FM1-43 fluorescence. These responses are too large to be caused by exocytosis. Instead, three lines of evidence suggest that FM1-43 is responding to phospholipid scrambling. First, ionomycin also stimulates increases in the fluorescence of annexin V, a phosphatidylserine-specific probe, while thapsigargin does not stimulate fluorescence increases of either probe. Secondly, cells that exhibit FM1-43 fluorescence increases after ionomycin stimulation stain with annexin V once FM1-43 is washed out. Thirdly, ionomycin stimulates uptake of 7-nitrobenz-2-oxa-1,3-diazole-labelled phosphatidylcholine, a specific assay for scramblase activity, whereas thapsigargin does not. We find that FM1-43 reports phospholipid scrambling with \'better\' kinetics than annexin V, and does require extracellular Ca(2+) to report phospholipid scrambling. We suggest that FM1-43 may be a useful probe to study the dynamics of phospholipid scrambling. The results are the first demonstration that FM1-43 can respond significantly to a biological process other than vesicular trafficking.