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Chris P M Reutelingsperger - One of the best experts on this subject based on the ideXlab platform.

  • visualization of cell death in mice with focal cerebral ischemia using fluorescent Annexin A5 propidium iodide and tunel staining
    Journal of Cerebral Blood Flow and Metabolism, 2011
    Co-Authors: Peyman Bahmani, Leo Hofstra, Eyk Schellenberger, Jan Klohs, Jens Steinbrink, Ryan Cordell, Marietta Zille, Jochen Muller, Denise Harhausen, Chris P M Reutelingsperger
    Abstract:

    To monitor stroke-induced brain damage and assess neuroprotective therapies, specific imaging of cell death after cerebral ischemia in a noninvasive manner is highly desirable. Annexin A5 has been suggested as a marker for imaging cell death under various disease conditions including stroke. In this study, C57BL6/N mice received middle cerebral artery occlusion (MCAO) and were injected intravenously with either active or inactive Cy5.5-Annexin A5 48 hours after reperfusion. Some mice also received propidium iodide (PI), a cell integrity marker. Only in mice receiving active Cy5.5-Annexin A5 were fluorescence intensities significantly higher over the hemisphere ipsilateral to MCAO than on the contralateral side. This was detected noninvasively and ex vivo 4 and 8 hours after injection. The majority of cells positive for fluorescent Annexin A5 were also positive for PI and fragmented DNA as detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL) staining. This study demonstrates the high specificity of Annexin A5 for visualization of cell death in a mouse model of stroke. To our knowledge, this is the first study to compare the distribution of injected active and inactive Annexin A5, PI, and TUNEL staining. It provides important information on the experimental and potential clinical applications of Annexin A5-based imaging agents in stroke.

  • extracellular Annexin A5 functions of phosphatidylserine binding and two dimensional crystallization
    Biochimica et Biophysica Acta, 2008
    Co-Authors: Hugo Van Genderen, Heidi Kenis, Leo Hofstra, Jagat Narula, Chris P M Reutelingsperger
    Abstract:

    In normal healthy cells phosphatidylserine is located in the inner leaflet of the plasma membrane. However, on activated platelets, dying cells and under specific circumstances also on various types of viable leukocytes phosphatidylserine is actively externalized to the outer leaflet of the plasma membrane. Annexin A5 has the ability to bind in a calcium-dependent manner to phosphatidylserine and to form a membrane-bound two-dimensional crystal lattice. Based on these abilities various functions for extracellular Annexin A5 on the phosphatidylserine-expressing plasma membrane have been proposed. In this review we describe possible mechanisms for externalization of Annexin A5 and various processes in which extracellular Annexin A5 may play a role such as blood coagulation, apoptosis, phagocytosis and formation of plasma membrane-derived microparticles. We further highlight the recent discovery of internalization of extracellular Annexin A5 by phosphatidylserine-expressing cells.

  • key role of platelet procoagulant activity in tissue factor and collagen dependent thrombus formation in arterioles and venules in vivo differential sensitivity to thrombin inhibition
    Microcirculation, 2008
    Co-Authors: Marijke J E Kuijpers, Imke C A Munnix, Judith M E M Cosemans, Bart J M Van Vlijmen, Mirjam Oude G A Egbrink, Chris P M Reutelingsperger, Johan W. M. Heemskerk
    Abstract:

    Objective: Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation.Methods: Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels.Results: Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI–mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled Annexin A5. Injection of Annexin A5 to shield exposed phos...

  • linking proteins with anionic nanoparticles via protamine ultrasmall protein coupled probes for magnetic resonance imaging of apoptosis
    Small, 2008
    Co-Authors: Eyk Schellenberger, Chris P M Reutelingsperger, Lisette Ungethum, Jorg Schnorr, Wolfdietrich Meyer, Matthias Taupitz, Bernd Hamm
    Abstract:

    Magnetic resonance imaging (MRI) of a target in vivo depends on the surface, size, and particle relaxivity of the target-specific nanoparticles for MRI. Here a new method for decorating very small iron oxide particles (VSOPs) with target-specific ligands is described. The method is based on the electrostatic attraction of the strongly positively charged peptide protamine to the anionic citrate shell of the electrostatically stabilized VSOPs. The protamine coat allows linkage chemistry and chimera technology to functionalize VSOPs or other negative charged surfaces with biologics. Annexin A5 (anxA5)-VSOP utilizing thiol chemistry was generated to couple biologically active anxA5 to VSOPs for in vivo MRI of apoptosis. Annexin A5-VSOP comprises five anxA5 molecules per iron oxide nanoparticle with a high R2 particle relaxivity of 180 000 mM(-1) s(-1) yet small hydrodynamic diameter of only 14.7+/-2.9 nm beneficial for in vivo MRI of extravascular targets.

  • factor xa driven thrombin generation in plasma dependency on the aminophospholipid density of membranes and inhibition by phospholipid binding proteins
    Thrombosis and Haemostasis, 2007
    Co-Authors: Simone J H Wielders, Chris P M Reutelingsperger, Edouard M Bevers, Lisette Ungethum, Theo Lindhout
    Abstract:

    Phosphatidylserine (PS) externalization of activated platelets plays a pivotal role in haemostasis and thrombosis. In the present study we have explored the relationship between the PS density of membranes and the rate of thrombin generation in plasma. Factor (F)Xa-initiated thrombin generation was measured in platelet-free plasma (PFP) containing either phospholipid vesicles of varying PS-content or non-stimulated platelets (reconstituted PRP).The duration of the initiation phase of FXa-driven thrombin generation decreased dramatically with increasing PS density. Concomitantly, the maximal rate of thrombin generation during the propagation phase (maxR) increased non-linearly, with the steepest incline between 5 and 10 mol% PS.Titration of FVa into plasma containing 2 mol% PS increased maxR proportionally and diminished the lag phase. In contrast, platelet-dependent thrombin generation was not influenced by addition of FVa.With increasing platelet concentration, the duration of the initiation phase drastically decreased, and maxR increased proportionally. At a physiologically relevant platelet concentration, maxR corresponded with the maxR found with 2 μM of 10 mol% PS. Annexin A5 (AnxA5) and lactadherin appeared to be powerful inhibitors of in-situ thrombin generation under all conditions examined, with AnxA5 being three- to fourfold more potent than lactadherin. In conclusion, maximal thrombin generation in plasma requires membranes with a density of 10–20 mol% PS. Our data further indicate that thrombin formed in situ induces externalization of PS to approx 10 mol% in a substantial platelet subpopulation.

Pascal Trouvé - One of the best experts on this subject based on the ideXlab platform.

  • Buserelin alleviates chloride transport defect in human cystic fibrosis nasal epithelial cells
    PLoS ONE, 2017
    Co-Authors: Marie-laure Calvez, Nathalie Benz, Florentin Huguet, Aude Saint-pierre, Elise Rouillé, Christelle Coraux, Claude Férec, Mathieu Kerbiriou, Pascal Trouvé
    Abstract:

    Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conduc-tance Regulator (CFTR) chloride (Cl-) channel regulated by protein kinases, phosphatases, divalent cations and by protein-protein interactions. Among protein-protein interactions, we previously showed that Annexin A5 (AnxA5) binds to CFTR and is involved in the channel localization within membranes and in its Cl-channel function. The deletion of phenylalanine at position 508 (F508del) is the most common mutation in CF which leads to an altered protein (F508del-CFTR) folding with a nascent protein retained within the ER and is quickly degraded. We previously showed that AnxA5 binds to F508del-CFTR and that its increased expression due to a Gonadoliberin (GnRH) augments Cl-efflux in cells expressing F508del-CFTR. The aim of the present work was to use the GnRH analog buserelin which is already used in medicine. Human nasal epithelial cells from controls and CF patients (F508del/ F508del) were treated with buserelin and we show here that the treatment alleviates Cl-channel defects in CF cells. Using proteomics we highlighted some proteins explaining this result. Finally, we propose that buserelin is a potential new pharmaceutical compound that can be used in CF and that bronchus can be targeted since we show here that they express GnRH-R.

Jacob H. Rand - One of the best experts on this subject based on the ideXlab platform.

  • inverted erythrocyte membranes demonstrate β2gpi antiphospholipid antibody interactions and membrane crosslinking
    Thrombosis Research, 2016
    Co-Authors: Saartje Bloemen, Jacob H. Rand, Bas De Laat, Katrien Devreese, Ljiljana V Vasovic
    Abstract:

    Abstract Introduction The antiphospholipid syndrome (APS) is an acquired autoimmune disorder predisposing patients to thrombosis or pregnancy complications. Since inverted erythrocyte membranes (iEMs) might provide a physiologically relevant source of anionic phospholipids, we studied the interactions of phospholipid-binding proteins and APS antibodies using iEMs. Materials & methods iEMs were prepared from packed erythrocytes by hypotonic lysis. Phosphatidylserine (PS) exposure was confirmed by Annexin A5 (A5) binding using fluorescence microscopy and flow cytometry. Binding of β2-glycoprotein I (β2GPI)–IgG immune complexes to iEMs was investigated with gel electrophoresis, western blot and flow cytometry. Functional involvement in coagulation was documented in the thrombin generation assay. Results iEMs readily precipitated purified β2GPI as well as β2GPI from normal plasma and APS plasma. The plasma of APS patients provided higher levels of IgG binding to iEMs relative to healthy controls. Thrombin generation increased with increasing concentrations of iEMs, documenting that coagulation proteins bound to the exposed phospholipids. The LA effect was also distinguished in thrombin generation when comparing APS patients, as indicated by an increased lag time. Agglutination was observed after incubation with APS patient plasma and this was augmented by anti-human globulin. Conclusions In conclusion, iEMs can provide a more physiological approach than phospholipid vesicle-based tests for investigating APS and are more amenable to standardization than platelet membranes.

  • Annexin A5 resistance is associated with thrombotic risk in patients with the lupus anticoagulant the vienna lupus anticoagulant and thrombosis study lats
    Blood, 2015
    Co-Authors: Florian Posch, Jacob H. Rand, Johanna Gebhart, Silvia Koder, Wu Xiaoxuan, Ingrid Pabinger
    Abstract:

    INTRODUCTION: Antiphospholipid (aPL) antibody mediated interference with the anticoagulant effect of Annexin A5 (A5) - designated "Annexin A5 resistance" (A5R) - has been implicated in adverse clinical outcomes. In this study, we ask whether A5R is associated with anamnestic thrombotic complications and adverse pregnancy outcomes in patients with the lupus anticoagulant (LA). PATIENTS & METHODS: We conducted a cross-sectional study including 143 patients (median age: 40.5 years, interquartile range (IQR): 31.8-60.0; female: n=119) who tested persistently positive for LA according to ISTH criteria (Miyakis et al. J Thromb Haemost 2006; 4: 295). Thrombotic complications were defined as a composite of arterial and/or venous thromboembolic events. Pregnancy complications were defined according to the modified Sapporo criteria. Out of the 143 patients, 94 (65.7%) had a history of a thrombembolic event (arterial: n=21, venous: n=80, both: n=7). Ninety-three women had at least one pregnancy, and 40 (43.0%) out of these women had at least one pregnancy complication as defined by the above mentioned criteria. It is important to mention that on average, these pregnancy complications had occurred more than ten years before study inclusion. A5R assays were done using phospholipid that was first exposed to patient plasmas as previously described; A5R results were expressed as the ratio of coagulation times with and without Annexin A5. In a subsample (n=109), a blinded external validation using two separate measurements of the A5R was performed. RESULTS: In blinded analysis, the concordance between the two A5R measurements was very high (Liu9s concordance correlation coefficient=0.83, average difference=12.2 units (95% limits of agreement from Bland-Altman analysis: -37.0 - 61.3). The A5R was inversely correlated with levels of antibodies against cardiolipin (Spearman9s rho for IgG and IgM: -0.65 and -0.24, respectively), and against beta2-glycoprotein I (Spearman9s rho for IgG and IgM: -0.57 and -0.28). The median A5R level was 205 units (IQR: 205-250, range: 130-336). Patients with a prior history of a thrombotic complications had significantly lower median levels of A5R (193.1 vs. 238.7 units, mean difference: 26.7 units (95% CI: 11.0-42.4), p=0.001). This difference was independent of both IgG and IgM isotype antibody levels against cardiolipin and beta2-glycoprotein I (adjusted mean difference: 20.7 units (95% CI: 6.4-35.0), p=0.005), and of whether patients were on anticoagulation or not (adjusted mean difference: 24.8 units (95% CI: 4.3 - 45.2), p=0.018). In the 95 women with at least one pregnancy, average A5R were lower in women with (n=40) compared to those without (n=53) at least one pregnancy complication (median: 200.9 vs. 231.9 units), however, this difference was not statistically significant (t-test p=0.255). CONCLUSION: A5R is associated with a history of thromboembolic complications in LA positive patients, suggesting that the ex vivo measurement of the A5R may reflect a prothrombotic phenotype in this patient population. Interestingly, we found no clear association between the A5R and anamnestic pregnancy complications was observed, however, the generalizability of this finding is hampered by the long time interval between anamnestic pregnancy complications and inclusion in this study. Disclosures No relevant conflicts of interest to declare.

  • hydroxychloroquine protects the Annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies evidence for a novel effect for an old antimalarial drug
    Blood, 2010
    Co-Authors: Jacob H. Rand, Anthony S Quinn, Pojen P Chen, Anthony W Ashton, James J Hathcock, Harry A M Andree, Douglas J Taatjes
    Abstract:

    Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

  • resistance to Annexin A5 anticoagulant activity a thrombogenic mechanism for the antiphospholipid syndrome
    Lupus, 2008
    Co-Authors: Jacob H. Rand, Anthony S Quinn, Douglas J Taatjes
    Abstract:

    The phospholipid binding protein, Annexin A5 (AnxA5), has potent anticoagulant properties that result from its forming 2-dimensional crystals over phospholipids, blocking the availability of the phospholipids for critical coagulation enzyme reactions. This article reviews the evidence that antiphospholipid antibodies can disrupt this anticoagulant shield and unmask thrombogenic anionic phospholipids, which may thereby contribute to thrombosis in patients with the antiphospholipid syndrome (APS). This mechanism for thrombosis in APS can be monitored with coagulation assays for resistance to anticoagulant activity of AnxA5.

  • correlation between antiphospholipid antibodies that recognize domain i of β2 glycoprotein i and a reduction in the anticoagulant activity of Annexin A5
    Blood, 2007
    Co-Authors: Bas De Laat, Menno Van Lummel, Ronald H W M Derksen, Philip G De Groot, Jacob H. Rand
    Abstract:

    The paradoxical correlation between thrombosis and the lupus anticoagulant (LAC) effect is an enigmatic feature of the antiphospholipid (aPL) syndrome. The Dutch authors previously reported that thrombosis-related anti-beta2-glycoprotein I (beta2GPI) antibodies recognize domain I and cause LAC. The American authors reported that aPLs disrupt an anticoagulant Annexin A5 (AnxA5) crystal shield. We investigated whether antidomain I antibodies correlate with disruption of AnxA5-anticoagulant activity. We studied a well-characterized group of 33 patients including subgroups with beta2GPI-dependent LAC that recognize domain I (n=11), with beta2GPI-independent LAC (n=12), and lacking LAC (n=10). The effects on AnxA5-anticoagulant activity were determined with an AnxA5 resistance assay that measures coagulation times with and without AnxA5. Patients with beta2GPI-dependent LAC (group A, all with thrombosis) had significantly lower AnxA5-anticoagulant ratios than those with beta2GPI-independent LAC (group B, thrombosis n=4; 157.8% versus 235.6%, P<.001) and those without LAC (group C, thrombosis n=2; 157.8% versus 232.5%, P<.001). There was no difference in the ratios between groups B and C (P=.92). Plasmas with beta2GPI-dependent LAC that recognize domain I displayed significantly increased AnxA5 resistance, suggesting that specifically anti-beta2GPI antibodies compete with AnxA5 for anionic phospholipids. These results are consistent with a model in which aPL antibodies may promote thrombosis by interfering with the anticoagulant activity of AnxA5.

Marie-laure Calvez - One of the best experts on this subject based on the ideXlab platform.

  • Buserelin alleviates chloride transport defect in human cystic fibrosis nasal epithelial cells
    PLoS ONE, 2017
    Co-Authors: Marie-laure Calvez, Nathalie Benz, Florentin Huguet, Aude Saint-pierre, Elise Rouillé, Christelle Coraux, Claude Férec, Mathieu Kerbiriou, Pascal Trouvé
    Abstract:

    Cystic fibrosis (CF) is the most common autosomal recessive disease in Caucasians caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conduc-tance Regulator (CFTR) chloride (Cl-) channel regulated by protein kinases, phosphatases, divalent cations and by protein-protein interactions. Among protein-protein interactions, we previously showed that Annexin A5 (AnxA5) binds to CFTR and is involved in the channel localization within membranes and in its Cl-channel function. The deletion of phenylalanine at position 508 (F508del) is the most common mutation in CF which leads to an altered protein (F508del-CFTR) folding with a nascent protein retained within the ER and is quickly degraded. We previously showed that AnxA5 binds to F508del-CFTR and that its increased expression due to a Gonadoliberin (GnRH) augments Cl-efflux in cells expressing F508del-CFTR. The aim of the present work was to use the GnRH analog buserelin which is already used in medicine. Human nasal epithelial cells from controls and CF patients (F508del/ F508del) were treated with buserelin and we show here that the treatment alleviates Cl-channel defects in CF cells. Using proteomics we highlighted some proteins explaining this result. Finally, we propose that buserelin is a potential new pharmaceutical compound that can be used in CF and that bronchus can be targeted since we show here that they express GnRH-R.

Nadja Bogdanova - One of the best experts on this subject based on the ideXlab platform.

  • maternal and paternal carriage of the Annexin A5 m2 haplotype a possible risk factor for recurrent implantation failure rif
    Journal of Assisted Reproduction and Genetics, 2021
    Co-Authors: N Rogenhofer, Arseni Markoff, Xenia Ennerst, Nadja Bogdanova, C J Thaler
    Abstract:

    OBJECTIVE This study was carried out to determine the potential role of the M2/ANXA5 haplotype as a risk factor for recurrent implantation failure (RIF). Carriage of the M2/ANXA5 haplotype that induces prothrombotic changes has been implicated in failure of early pregnancies and placenta-mediated complications (preeclampsia, IUGR, preterm birth). MATERIAL AND METHODS In the present case control study, 63 couples (females and males) with RIF presenting for IVF/ICSI to the Fertility Center of [masked] were analyzed. RIF was defined as ≥ 4 consecutive failed ART-transfers of ≥ 4 blastocysts or ≥ 8 cleavage-stage embryos of optimal quality and maternal age ≤ 41. Fertile female controls (n = 90) were recruited from the same center. Population controls (n = 533) were drafted from the PopGen biobank, UKSH Kiel. RESULTS Couples carrying the M2/ANXA5 haplotype turned out to have a significantly increased relative risk (RR) for RIF. Compared with female fertile controls, RR was 1.81 with p = 0.037 (OR 2.1, 95%CI 1.0-4.3) and RR was 1.70, with p = 0.004 (OR 2.0, 95%CI 1.2-3.1) compared with population controls (15.4% M2 carriers). Male partners were comparable with RIF females for M2/ANXA5 haplotypes (28.6% vs. 23.8%, p = 0.54). RIF females compared with population controls had a RR of 1.55 (p = 0.09) and RIF males compared with population controls had a RR of 1.9 (p = 0.01). Couples with ≥ 7 failed transfers showed a RR of 1.82 (p = 0.02) compared with population controls. CONCLUSION Our findings suggest that maternal as well as paternal M2/ANXA5 haplotype carriages are risk factors for RIF. These results allow new insights into the pathogenesis of RIF and might help to identify relevant risk groups.

  • Further insights into the role of the Annexin A5 M2 haplotype as recurrent pregnancy loss factor, assessing timing of miscarriage and partner risk.
    Fertility and Sterility, 2013
    Co-Authors: Frank Tuttelmann, Arseni Markoff, P Ivanov, Charlotte Dietzel, Anna Sofroniou, Tsvetomira M. Tsvyatkovska, Regina Komsa-penkova, Peter Wieacker, Nadja Bogdanova
    Abstract:

    Objective To study the influence of M2/ANXA5 for recurrent pregnancy loss (RPL), according to the timing of miscarriages and assess the male partner risk. Design Genetic association study. Setting Academic research center. Patient(s) Female patients from two academic centers in Germany and Bulgaria with two or more unexplained miscarriages were selected for this study. Male partners were available for a part of the German sample. Population controls were recruited from healthy individuals of respective populations. Intervention(s) None. Main Outcome Measure(s) Incidence of M2 carriage and odds ratios were calculated between patient and control groups, and RPL risk was evaluated. Result(s) The M2 haplotype in ANXA5 was associated with greater overall RPL risk in German and in Bulgarian women, and a trend of higher prevalence was seen for male partners of German RPL patients. The highest relative risk of M2 carriage was observed in women of both populations with "early" fetal losses between the 10th and 15th gestational weeks, which was significant in the meta-analysis. Conclusion(s) M2 carriage seems to have an RPL risk role mostly for early abortions, gestational weeks 10–15. In the first phase of pregnancy this correlates with vascular remodeling to accomplish the transition from high- to low-resistance blood vessels.

  • paternal and maternal carriage of the Annexin A5 m2 haplotype are equal risk factors for recurrent pregnancy loss a pilot study
    Fertility and Sterility, 2012
    Co-Authors: N Rogenhofer, Arseni Markoff, Nadja Bogdanova, Laura Engels, Frank Tuttelmann, C J Thaler
    Abstract:

    Objective To study the possible contribution of paternal, in addition to maternal, carriage of M2/ANXA5 as a risk factor for recurrent pregnancy loss (RPL). Design Case–control study. Setting Academic research center. Patient(s) Couples presenting themselves to the Fertility Center, Ludwig-Maximilians-University Munich with two or more consecutive, unexplained miscarriages were selected for this study. Fertile female controls were from the same center and also from the resource of the Institute of Human Genetics, Westfalian Wilhelms-University Muenster. Population controls were drafted from the PopGen biobank, University Clinic Schleswig-Holstein Kiel. Intervention(s) None. Main Outcome Measure(s) Incidence of M2 carriage was estimated in patient and control groups, odds ratios were calculated, and RPL risk was evaluated. Result(s) In comparison with female fertile controls, the risk for repeated abortion in the RPL group, associated with M2 carriage, was between 1.7 and 3.8, and it was 2.3 compared with population controls. Because of the equal genetic incidence of M2, with an allelic frequency of 0.167 in the female and male partner RPL subgroups, the haplotype confers approximately the same relative risk to carriers of both sexes. Conclusion(s) Paternal M2 carriage seems to confer an equal risk for recurrent miscarriages as M2 carriage in RPL mothers. This finding points to a role of ANXA5 and the M2 haplotype in the fetus and/or the extraembryonic membranes for pregnancy pathology. Prognostic RPL algorithms might be improved by testing the male partner for M2 carriage, and this may guide adequate therapeutic decisions.